Dosages for treatment with anti-ErbB2 antibodies

ABSTRACT

The present invention concerns the treatment of disorders characterized by the overexpression of ErbB2. More specifically, the invention concerns the treatment of human patients susceptible to or diagnosed with cancer overexpressing ErbB2 with anti-ErbB2 antibody.

RELATED APPLICATIONS

This application is divisional of U.S. Ser. No. 09/648,067 filed Aug.25, 2000 (now U.S. Pat. No. 6,627,196), which claims priority under 35USC 119(e) to provisional application Nos. 60/151,018, filed Aug. 27,1999 and 60/213,822, filed Jun. 23, 2000, the contents of which areincorporated heroin by reference.

FIELD OF THE INVENTION

The present invention concerns the treatment of disorders characterizedby the overexpression of ErbB2 or disorders expressing epidermal growthfactor receptor (EGFR), comprising administering to a human or animalpresenting the disorders a therapeutically effective amount of anantibody that binds ErbB2. More specifically, the invention concerns thetreatment of human patients susceptible to or diagnosed with canceroverexpressing ErbB2 or expressing EGFR, where the treatment is with ananti-ErbB2 antibody administered by front loading the dose of antibodyduring treatment by intravenous and/or subcutaneous administration. Theinvention optionally includes treatment of cancer in a human patientwith a combination of an anti-ErbB2 antibody and a chemotherapeuticagent, such as, but not limited to, a taxoid. The taxoid may be, but isnot limited to paclitaxel or docetaxel. The invention further includestreatment of cancer in a human patient with a combination of anti-ErbB2antibody and a chemotherapeutic agent, such as, but not limited to, ananthracycline derivative. Optionally, treatment with a combination ofanti-ErbB2 and an anthracycline derivative includes treatment with aneffective amount of a cardioprotectant. The present invention furtherconcerns infrequent dosing of anti-ErbB2 antibodies.

BACKGROUND OF THE INVENTION

Proto-oncogenes that encode growth factors and growth factor receptorshave been identified to play important roles in the pathogenesis ofvarious human malignancies, including breast cancer. It has been foundthat the human ErbB2 gene (erbB2, also known as her2, or c-erbB-2),which encodes a 185-kd transmembrane glycoprotein receptor (p185^(HER2))related to the epidermal growth factor receptor (EGFR), is overexpressedin about 25% to 30% of human breast cancer (Slamon et al., Science235:177-182 [1987]; Slamon et al., Science 244:707-712 [1989]).

Several lines of evidence support a direct role for ErbB2 in thepathogenesis and clinical aggressiveness of ErbB2-overexpressing tumors.The introduction of ErbB2 into non-neoplastic cells has been shown tocause their malignant transformation (Hudziak et al., Proc. Natl. Acad.Sci. USA 84:7159-7163 [1987]; DiFiore et al., Science 237:78-182[1987]). Transgenic mice that express HER2 were found to develop mammarytumors (Guy et al., Proc. Natl. Acad. Sci. USA 89:10578-10582 [1992]).

Antibodies directed against human erbB2 protein products and proteinsencoded by the rat equivalent of the erbB2 gene (neu) have beendescribed. Drebin et al., Cell 41:695-706 (1985) refer to an IgG2amonoclonal antibody which is directed against the rat neu gene product.This antibody called 7.16.4 causes down-modulation of cell surface p185expression on B104-1-1 cells (NIH-3T3 cells transfected with the neuproto-oncogene) a inhibits colony formation of these cells. In Drebin etal. PNAS (USA) 83:9129-9133 (1986), the 7.16.4 antibody was shown toinhibit the tumorigenic growth of neu-transformed NIH-3T3 cells as wellas rat neuroblastoma cells (from which the neu oncogene was initiallyisolated) implanted into nude mice. Drebin et al. in Oncogene 2:387-394(1988) discuss the production of a panel of antibodies against the ratneu gene product. Al of the antibodies were found to exert a cytostaticeffect on the growth of neu-transformed cells suspended in soft agar.Antibodies of the IgM, IgG2a and IgG2b isotypes were able to mediatesignificant in vitro lysis of neu-transformed cells in the presence ofcomplement, whereas none of the antibodies were able to mediate highlevels of antibody-dependent cellular cytotoxicity (ADCC) of theneu-transformed cells. Drebin et al. Oncogene 2:273-277 (1988) reportthat mixtures of antibodies reactive with two distinct regions on thep185 molecule result in synergistic anti-tumor effects onneu-transformed NIH-3T3 cells implanted into nude mice. Biologicaleffects of anti-neu antibodies are reviewed in Myers et al., Meth.Enzym. 198:277-290 (1991). See also WO94/22478 published Oct. 13, 1994.Hudziak et al., Mol. Cell. Biol. 9(3): 1165-1172 (1989) describe thegeneration of a panel of anti-ErbB2 antibodies which were characterizedusing the human breast tumor cell line SKBR3. Relative cellproliferation of the SKBR3 cells following exposure to the antibodieswas determined by crystal violet staining of the monolayers after 72hours. Using this assay, maximum inhibition was obtained with theantibody called 4D5 which inhibited cellular proliferation by 56%. Otherantibodies in the panel, including 7C2 and 7F3, reduced cellularproliferation to a lesser extent in this assay. Hudziak et al. concludethat the effect of the 4D5 antibody on SKBR3 cells was cytostatic ratherthan cytotoxic, since SKBR3 cells resumed growth at a nearly normal ratefollowing removal of the antibody from the medium. The antibody 4D5 wasfurther found to sensitize p185^(erbB2)-overexpressing breast tumor celllines to the cytotoxic effects of TNF-α. See also WO89/06692 publishedJul. 27, 1989. The anti-ErbB2 antibodies discussed in Hudziak et al. arefurther characterized in Fendly et al. Cancer Research 50:1550-1558(1990); Kotts et al. In Vitro 26(3):59A (1990); Sarup et al. GrowthRegulation 1:72-82 (1991); Shepard et al. J. Clin. Immunol.11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol. 11(2):979-986(1991); Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993);Pietras et al. Oncogene 9:1829-1838 (1994); Vitetta et al. CancerResearch 54:5301-5309 (1994); Sliwkowski et al. J. Biol. Chem.269(20):14661-14665 (1994); Scott et al. J. Biol. Chem. 266:14300-5(1991); and D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994).

Tagliabue et al. Int. J. Cancer 47:933-937 (1991) describe twoantibodies which were selected for their reactivity on the lungadenocarcinoma cell line (Calu-3) which overexpresses ErbB2. One of theantibodies, called MGR3, was found to internalize, inducephosphorylation of ErbB2, and inhibit tumor cell growth in vitro.

McKenzie et al. Oncogene 4:543-548 (1989) generated a panel ofanti-ErbB2 antibodies with varying epitope specificities, including theantibody designated TA1. This TA1 antibody was found to induceaccelerated endocytosis of ErbB2 (see Maier et al. Cancer Res.51:5361-5369 [1991]). Bacus et al. Molecular Carcinogenesis 3:350-362(1990) reported that the TA1 antibody induced maturation of the breastcancer cell lines AU-565 (which overexpresses the erbB2 gene) and MCF-7(which does not). Inhibition of growth and acquisition of a maturephenotype in these cells was found to be associated with reduced levelsof ErbB2 receptor at the cell surface and transient increased levels inthe cytoplasm.

Stancovski et al. PNAS (USA) 88:8691-8695 (1991) generated a panel ofanti-ErbB2 antibodies, injected them i.p. into nude mice and evaluatedtheir effect on tumor growth of murine fibroblasts transformed byoverexpression of the erbB2 gene. Various levels of tumor inhibitionwere detected for four of the antibodies, but one of the antibodies(N28) consistently stimulated tumor growth. Monoclonal antibody N28induced significant phosphorylation of the ErbB2 receptor, whereas theother four antibodies generally displayed low or nophosphorylation-inducing activity. The effect of the anti-ErbB2antibodies on proliferation of SKBR3 cells was also assessed. In thisSKBR3 cell proliferation assay, two of the antibodies (N12 and N29)caused a reduction in cell proliferation relative to control. Theability of the various antibodies to induce cell lysis in vitro viacomplement-dependent cytotoxicity (CDC) and antibody-mediatedcell-dependent cytotoxicity (ADCC) was assessed, with the authors ofthis paper concluding that the inhibitory function of the antibodies wasnot attributed significantly to CDC or ADCC.

Bacus et al. Cancer Research 52:2580-2589 (1992) further characterizedthe antibodies described in Bacus et al. (1990) and Stancovski et al. ofthe preceding paragraphs. Extending the i.p. studies of Stancovski etal., the effect of the antibodies after i.v. injection into nude miceharboring mouse fibroblasts overexpressing human ErbB2 was assessed. Asobserved in their earlier work, N28 accelerated tumor growth, whereasN12 and N29 significantly inhibited growth of the ErbB2-expressingcells. Partial tumor inhibition was also observed with the N24 antibody.Bacus et al. also tested the ability of the antibodies to promote amature phenotype in the human breast cancer cell lines AU-565 andMDA-MB453 (which overexpress ErbB2) as well as MCF-7 (containing lowlevels of the receptor). Bacus et al. saw a correlation between tumorinhibition in vivo and cellular differentiation; the tumor-stimulatoryantibody N28 had no effect on differentiation, and the tumor inhibitoryaction of the N12, N29 and N24 antibodies correlated with the extent ofdifferentiation they induced.

Xu et al. Int. J. Cancer 53:401-408 (1993) evaluated a panel ofanti-ErbB2 antibodies for their epitope binding specificities, as wellas their ability to inhibit anchorage-independent andanchorage-dependent growth of SKBR3 cells (by individual antibodies andin combinations), modulate cell-surface ErbB2, and inhibit ligandstimulated anchorage-independent growth. See also WO94/00136 publishedJan. 6, 1994 and Kasprzyk et al. Cancer Research 52:2771-2776 (1992)concerning anti-ErbB2 antibody combinations. Other anti-ErbB2 antibodiesare discussed in Hancock et al. Cancer Res. 51:4575-4580 (1991); Shawveret al. Cancer Res. 54:1367-1373 (1994); Arteaga et al. Cancer Res.54:3758-3765 (1994); and Harwerth et al. J. Biol. Chem. 267:15160-15167(1992).

A recombinant humanized anti-ErbB2 monoclonal antibody (a humanizedversion of the murine anti-ErbB2 antibody 4D5, referred to as rhuMAbHER2, HERCEPTIN®, or HERCEPTIN® anti-ErbB2 antibody) has been clinicallyactive in patients with ErbB2-overexpressing metastatic breast cancersthat had received extensive prior anti-cancer therapy (Baselga et al.,J. Clin. Oncol. 14:737-744 [1996]). The recommended initial loading dosefor HERCEPTIN® is 4 mg/kg administered as a 90-minute infusion. Therecommended weekly maintenance dose is 2 mg/kg and can be administeredas a 30-minute infusion if the initial loading dose is well tolerated.

ErbB2 overexpression is commonly regarded as a predictor of a poorprognosis, especially in patients with primary disease that involvesaxillary lymph nodes (Slamon et al., [1987] and [1989], supra; Ravdinand Chamness, Gene 159:19-27 [1995]; and Hynes and Stern, BiochimBiophys Acta 1198:165-184 [1994]), and has been linked to sensitivityand/or resistance to hormone therapy and chemotherapeutic regimens,including CMF (cyclophosphamide, methotrexate, and fluoruracil) andanthracyclines (Baselga et al., Oncology 11(3 Supp11):43-48 [1997]).However, despite the association of ErbB2 overexpression with poorprognosis, the odds of HER2-positive patients responding clinically totreatment with taxanes were greater than three times those ofHER2-negative patients (Ibid). rhuMab HER2 was shown to enhance theactivity of paclitaxel (TAXOL®) and doxorubicin against breast cancerxenografts in nude mice injected with BT-474 human breast adenocarcinomacells, which express high levels of HER2 (Baselga et al., Breast Cancer,Proceedings of ASCO, Vol. 13, Abstract 53 [1994]).

SUMMARY OF THE INVENTION

The present invention concerns the discovery that an early attainment ofan efficacious target trough serum concentration by providing an initialdose or doses of anti-ErbB2 antibodies followed by subsequent doses ofequal or smaller amounts of antibody (greater front loading) is moreefficacious than conventional treatments. The efficacious target troughserum concentration is reached in 4 weeks or less, preferably 3 weeks orless, more preferably 2 weeks or less, and most preferably 1 week orless, including 1 day or less. The target serum concentration isthereafter maintained by the administration of maintenance doses ofequal or smaller amounts for the remainder of the treatment regimen oruntil suppression of disease symptoms is achieved.

The invention further concerns a method for the treatment of a humanpatient susceptible to or diagnosed with a disorder characterized byoverexpression of ErbB2 receptor comprising administering atherapeutically effective amount of an anti-ErbB2 antibodysubcutaneously. Preferably, the initial dose (or doses) as well as thesubsequent maintenance dose or doses are administered subcutaneously.Optionally, where the patient's tolerance to the anti-ErbB2 antibody isunknown, the initial dose is administered by intravenous infusion,followed by subcutaneous administration of the maintenance doses if thepatient's tolerance for the antibody is acceptable.

According to the invention, the method of treatment involvesadministration of an initial dose of anti-ErbB2 antibody of more thanapproximately 4 mg/kg, preferably more than approximately 5 mg/kg. Themaximum initial dose or a subsequent dose does not exceed 50 mg/kg,preferably does not exceed 40 mg/kg, and more preferably does not exceed30 mg/kg. Administration is by intravenous or subcutaneousadministration, preferably intravenous infusion or bolus injection, ormore preferably subcutaneous bolus injection. The initial dose may beone or more administrations of drug sufficient to reach the targettrough serum concentration in 4 weeks or less, preferably 3 weeks orless, more preferably 2 weeks or less, and most preferably 1 week orless, including one day or less.

According to the invention, the initial dose or doses is/are followed bysubsequent doses of equal or smaller amounts of antibody at intervalssufficiently close to maintain the trough serum concentration ofantibody at or above an efficacious target level. Preferably, an initialdose or subsequent dose does not exceed 50 mg/kg, and each subsequentdose is at least 0.01 mg/kg. Preferably the amount of drug administeredis sufficient to maintain the target trough serum concentration suchthat the interval between administration cycles is at least one week.Preferably the trough serum concentration does not exceed 2500 μg/ml anddoes not fall below 0.01 μg/ml during treatment. The front loading drugtreatment method of the invention has the advantage of increasedefficacy by reaching a target serum drug concentration early intreatment. The subcutaneous delivery of maintenance doses according tothe invention has the advantage of being convenient for the patient andhealth care professionals, reducing time and costs for drug treatment.Preferably, the initial dose (or the last dose within an initial doseseries) is separated in time from the first subsequent dose by 4 weeksor less, preferably 3 weeks or less, more preferably 3 weeks or less,most preferably 1 week or less.

In an embodiment of the invention, the initial dose of anti-ErbB2 is 6mg/kg, 8 mg/kg, or 12 mg/kg delivered by intravenous or subcutaneousadministration, such as intravenous infusion or subcutaneous bolusinjection. The subsequent maintenance doses are 2 mg/kg delivered onceper week by intravenous infusion, intravenous bolus injection,subcutaneous infusion, or subcutaneous bolus injection. The choice ofdelivery method for the initial and maintenance doses is made accordingto the ability of the animal or human patient to tolerate introductionof the antibody into the body. Where the antibody is well-tolerated, thetime of infusion may be reduced. The choice of delivery method asdisclosed for this embodiment applies to all drug delivery regimenscontemplated according to the invention.

In another embodiment, the invention includes an initial dose of 12mg/kg anti-ErbB2 antibody, followed by subsequent maintenance doses of 6mg/kg once per 3 weeks.

In still another embodiment, the invention includes an initial dose of 8mg/kg anti-ErbB2 antibody, followed by 6 mg/kg once per 3 weeks.

In yet another embodiment, the invention includes an initial dose of 8mg/kg anti-ErbB2 antibody, followed by subsequent maintenance doses of 8mg/kg once per week or 8 mg/kg once every 2 to 3 weeks.

In another embodiment, the invention includes initial doses of at least1 mg/kg, preferably 4 mg/kg, anti-ErbB2 antibody on each of days 1, 2and 3, followed by subsequent maintenance doses of 6 mg/kg once per 3weeks.

In another embodiment, the invention includes an initial dose of 4 mg/kganti-ErbB2 antibody, followed by subsequent maintenance doses of 2 mg/kgtwice per week, wherein the maintenance doses are separated by 3 days.

In still another embodiment, the invention includes a cycle of dosing inwhich delivery of anti-ErbB2 antibody is 2-3 times per week for 3 weeks.In one embodiment of the invention, each dose is approximately 25 mg/kgor less for a human patient, preferably approximately 10 mg/kg or less.This 3 week cycle is preferably repeated as necessary to achievesuppression of disease symptoms.

In another embodiment, the invention includes a cycle of dosing in whichdelivery of anti-ErbB2 antibody is daily for 5 days. According to theinvention, the cycle is preferably repeated as necessary to achievesuppression of disease symptoms.

The disorder preferably is a benign or malignant tumor characterized bythe overexpression of the ErbB2 receptor, e.g. a cancer, such as, breastcancer, squamous cell cancer, small-cell lung cancer, non-small celllung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,colon cancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer. The method of the invention may further comprise administrationof a chemotherapeutic agent other than an anthracycline, e.g.doxorubicin or epirubicin. The chemotherapeutic agent preferably is ataxoid, such as TAXOL® (paclitaxel) or a TAXOL® derivative.

Preferred anti-ErbB2 antibodies bind the extracellular domain of theErbB2 receptor, and preferably bind to the epitope 4D5 or 3H4 within theErbB2 extracellular domain sequence. More preferably, the antibody isthe antibody 4D5, most preferably in a humanized form. Other preferredErbB2-binding antibodies include, but are not limited to, antibodies7C2, 7F3, and 2C4, preferably in a humanized form.

The method of the present invention is particularly suitable for thetreatment of breast or ovarian cancer, characterized by theoverexpression of the ErbB2 receptor.

The present application also provides a method of therapy involvinginfrequent dosing of an anti-ErbB2 antibody. In particular, theinvention provides a method for the treatment of cancer (e.g. cancercharacterized by overexpression of the ErbB2 receptor) in a humanpatient comprising administering to the patient a first dose of ananti-ErbB2 antibody followed by at least one subsequent dose of theantibody, wherein the first dose and subsequent dose are separated fromeach other in time by at least about two weeks (e.g. from about twoweeks to about two months), and optionally at least about three weeks(e.g. from about three weeks to about six weeks). For instance, theantibody may be administered about every three weeks, about two to about20 times, e.g. about six times. The first dose and subsequent dose mayeach be from about 2 mg/kg to about 16 mg/kg; e.g. from about 4 mg/kg toabout 12 mg/kg; and optionally from about 6 mg/kg to about 12 mg/kg.Generally, two or more subsequent doses (e.g. from about two to aboutten subsequent doses) of the antibody are administered to the patient,and those subsequent doses are preferably separated from each other intime by at least about two weeks (e.g. from about two weeks to about twomonths), and optionally at least about three weeks (e.g. from aboutthree weeks to about six weeks). The two or more subsequent doses mayeach be from about 2 mg/kg to about 16 mg/kg; or from about 4 mg/kg toabout 12 mg/kg; or from about 6 mg/kg to about 12 mg/kg. The inventionadditionally provides an article of manufacture, comprising a container,a composition within the container comprising an anti-ErbB2 antibody,and a package insert containing instructions to administer the antibodyaccording to such methods.

The presently described dosing protocols may be applied to otheranti-ErbB antibodies such as anti-epidermal growth factor receptor(EGFR), anti-ErbB3 and anti-ErbB4 antibodies. Thus, the inventionprovides a method for the treatment of cancer in a human patient,comprising administering an effective amount of an anti-ErbB antibody tothe human patient, the method comprising administering to the patient aninitial dose of at least approximately 5 mg/kg of the anti-ErbBantibody; and administering to the patient a plurality of subsequentdoses of the antibody in an amount that is approximately the same orless than the initial dose. Alternatively, or additionally, theinvention pertains to a method for the treatment of cancer in a humanpatient comprising administering to the patient a first dose of ananti-ErbB antibody followed by at least one subsequent dose of theantibody, wherein the first dose and subsequent dose are separated fromeach other in time by at least about two weeks. The inventionadditionally provides an article of manufacture, comprising a container,a composition within the container comprising an anti-ErbB antibody, anda package insert containing instructions to administer the antibodyaccording to such methods.

In another aspect, the invention concerns an article of manufacture,comprising a container, a composition within the container comprising ananti-ErbB2 antibody, optionally a label on or associated with thecontainer that indicates that the composition can be used for treating acondition characterized by overexpression of ErbB2 receptor, and apackage insert containing instructions to avoid the use ofanthracycline-type chemotherapeutics in combination with thecomposition. According to the invention, the package insert furtherincludes instructions to administer the anti-ErbB2 antibody at aninitial dose of 5 mg/kg followed by the same or smaller subsequent doseor doses. In another embodiment of the invention, the package insertfurther includes instructions to administer the anti-ErbB2 antibodysubcutaneously for at least one of the doses, preferably for all of thesubsequent doses following the initial dose, most preferably for alldoses.

In a further aspect, the invention provides a method of treating ErbB2expressing cancer in a human patient comprising administering to thepatient effective amounts of an anti-ErbB2 antibody and achemotherapeutic agent. In one embodiment of the invention, thechemotherapeutic agent is a taxoid including, but not limited to,paclitaxel and docetaxel. In another embodiment, the chemotherapeuticagent is an anthracyline derivative including, but not limited to,doxorubicin or epirubicin. In still another embodiment of the invention,treatment with an anti-ErbB2 antibody and an anthracycline derivativefurther includes administration of a cardioprotectant to the patient. Instill another embodiment, an anthracycline derivative is notadministered to the patient with the anti-ErbB2 antibody. One or moreadditional chemotherapeutic agents may also be administered to thepatient. The cancer is preferably characterized by overexpression ofErbB2.

The invention further provides an article of manufacture comprising acontainer, a composition within the container comprising an anti-ErbB2antibody and a package insert instructing the user of the composition toadminister the anti-ErbB2 antibody composition and a chemotherapeuticagent to a patient. In another embodiment, the chemotherapeutic agent isother than an anthracycline, and is preferably a taxoid, such as TAXOL®.In still another embodiment, the chemotherapeutic agent is ananthracycline, including but not limited to, doxorubicin or epirubicin.In yet another embodiment, the chemotherapeutic agent is ananthracycline and the package insert further instructs the user toadminister a cardioprotectant.

The methods and compositions of the invention comprise an anti-ErbB2antibody and include a humanized anti-ErbB2 antibody. Thus, theinvention further pertains to a composition comprising an antibody thatbinds ErbB2 and the use of the antibody for treating ErbB2 expressingcancer, e.g., ErbB2 overexpressing cancer, in a human. The inventionalso pertains to the use of the antibody for treating EGFR expressingcancer. Preferably the antibody is a monoclonal antibody 4D5, e.g.,humanized 4D5 (and preferably huMAb4D5-8 (HERCEPTIN® anti-ErbB2antibody); or monoclonal antibody 2C4, e.g., humanized 2C4. The antibodymay be an intact antibody (e.g., an intact IgG₁ antibody) or an antibodyfragment (e.g., a Fab, F(ab′)₂, diabody, and the like). The variablelight chain and variable heavy chain regions of humanized anti-ErbB2antibody 2C4 are shown in FIGS. 5A and 5B.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows epitope-mapping of the extracellular domain of ErbB2 asdetermined by truncation mutant analysis and site-directed mutagenesis(Nakamura et al. J. of Virology 67(10):6179-6191 [October 1993]; Renz etal. J. Cell Biol. 125(6):1395-1406 [June 1994]). The anti-proliferativeMabs 4D5 and 3H4 bind adjacent to the transmembrane domain. The variousErbB2-ECD truncations or point mutations were prepared from cDNA usingpolymerase chain reaction technology. The ErbB2 mutants were expressedas gD fusion proteins in a mammalian expression plasmid. This expressionplasmid uses the cytomegalovirus promoter/enhancer with SV40 terminationand polyadenylation signals located downstream of the inserted cDNA.Plasmid DNA was transfected into 293S cells. One day followingtransfection, the cells were metabolically labeled overnight inmethionine and cysteine-free, low glucose DMEM containing 1% dialyzedfetal bovine serum and 25 μCi each of ³⁵S methionine and ³⁵S cysteine.Supernatants were harvested either the ErbB2 MAbs or control antibodieswere added to the supernatant and incubated 24 hours at 4° C. Thecomplexes were precipitated, applied to a 10-20% Tricine SDS gradientgel and electrophoresed at 100 V. The gel was electroblotted onto amembrane and analyzed by autoradiography. SEQ ID NOs:8 and 9 depict the3H4 and 4D5 epitopes, respectively.

FIG. 2 depicts with underlining the amino acid sequence of Domain 1 ofErbB2 (SEQ ID NO:1). Bold amino acids indicate the location of theepitope recognized by MAbs 7C2 and 7F3 as determined by deletionmapping, i.e. the “7C2/7F3 epitope” (SEQ ID NO:2).

FIG. 3 is a graph of anti-ErbB2 antibody (HERCEPTIN®) trough serumconcentration (μg/ml, mean±SE, dark circles) by week from week 2 throughweek 36 for ErbB2 overexpressing patients treated with HERCEPTIN®anti-ErbB2 antibody at 4 mg/kg initial dose, followed by 2 mg/kg weekly.The number of patients at each time point is represented by “n” (whitesquares).

FIG. 4A is a linear plot of tumor volume changes over time in micetreated with HERCEPTIN® anti-ErbB2 antibody. FIG. 4B is asemi-logarithmic plot of the same data as in FIG. 4A such that thevariation in tumor volume for the treated animals is observed morereadily.

FIGS. 5A and 5B depict alignments of the amino acid sequences of thevariable light (V_(L)) (FIG. 5A) and variable heavy (V_(H)) (FIG. 5B)domains of murine monoclonal antibody 2C4 (SEQ ID Nos. 10 and 11,respectively); V_(L) and V_(H) domains of humanized Fab version 574 (SEQID Nos. 12 and 13, respectively), and human V_(L) and V_(H) consensusframeworks (hum κ1, light kappa subgroup I; humIII, heavy subgroup III)(SEQ ID Nos. 14 and 15, respectively). Asterisks identify differencesbetween humanized Fab version 574 and murine monoclonal antibody 2C4 orbetween humanized Fab version 574 and the human framework.Complementarity Determining Regions (CDRs) are in brackets. HumanizedFab version 574, with the changes ArgH71Val, AspH73Arg and IleH69Leu,appears to have binding restored to that of the original chimeric 2C4Fab fragment. Additional FR and/or CDR residues, such as L2, L54, L55,L56, H35 and/or H48, may be modified (e.g. substituted asfollows—IleL2Thr; ArgL54Leu; TyrL55Glu; ThrL56Ser; AspH35Ser; andValH48Ile) in order to further refine or enhance binding of thehumanized antibody. Alternatively, or additionally, the humanizedantibody may be affinity matured in order to further improve or refineits affinity and/or other biological activities.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

An “ErbB receptor” is a receptor protein tyrosine kinase which belongsto the ErbB receptor family and includes EGFR, HER2, ErbB3 and ErbB4receptors as well as TEGFR (U.S. Pat. No. 5,708,156) and other membersof this family to be identified in the future. The ErbB receptor willgenerally comprise an extracellular domain, which may bind an ErbBligand; a lipophilic transmembrane domain; a conserved intracellulartyrosine kinase domain; and a carboxyl-terminal signaling domainharboring several tyrosine residues which can be phosphorylated. TheErbB receptor may be a native sequence ErbB receptor or an amino acidsequence variant thereof. Preferably the ErbB receptor is nativesequence human ErbB receptor.

The terms “ErbB1”, “epidermal growth factor receptor” and “EGFR” areused interchangeably herein and refer to native sequence EGFR asdisclosed, for example, in Carpenter et al. Ann. Rev. Biochem.56:881-914 (1987), including variants thereof (e.g. a deletion mutantEGFR as in Humphrey et al. PNAS (USA) 87:4207-4211 (1990)). erbB1 refersto the gene encoding the EGFR protein product. Examples of antibodieswhich bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRLHB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S.Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such aschimerized 225 (C225) and reshaped human 225 (H225) (see, WO 96/40210,Imclone Systems Inc.).

“ErbB3” and “HER3” refer to the receptor polypeptide as disclosed, forexample, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus etal. PNAS (USA) 86:9193-9197 (1989), including variants thereof Examplesof antibodies which bind HER3 are described in U.S. Pat. No. 5,968,511(Akita and Sliwkowski), e.g. the 8B8 antibody (ATCC HB 12070) or ahumanized variant thereof

The terms “ErbB4” and “HER4” herein refer to the receptor polypeptide asdisclosed, for example, in EP Pat Appln No 599,274; Plowman et al.,Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al.,Nature, 366:473-475 (1993), including variants thereof such as the HER4isoforms disclosed in WO 99/19488.

The terms “HER2”, “ErbB2” “c-Erb-B2” are used interchangeably. Unlessindicated otherwise, the terms “ErbB2” “c-Erb-B2” and “HER2” when usedherein refer to the human protein, and “erbB2,” “c-erb-B2,” and “her2”refer to human gene. The human erbB2 gene and ErbB2 protein are, forexample, described in Semba et al., PNAS (USA) 82:6497-6501 (1985) andYamamoto et al. Nature 319:230-234 (1986) (Genebank accession numberX03363). ErbB2 comprises four domains (Domains 1-4).

The “epitope 4D5” is the region in the extracellular domain of ErbB2 towhich the antibody 4D5 (ATCC CRL 10463) binds. This epitope is close tothe transmembrane region of ErbB2. To screen for antibodies which bindto the 4D5 epitope, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed (see FIG. 1) to assesswhether the antibody binds to the 4D5 epitope of ErbB2 (i.e. any one ormore residues in the region from about residue 529, e.g. about residue561 to about residue 625, inclusive).

The “epitope 3H4” is the region in the extracellular domain of ErbB2 towhich the antibody 3H4 binds. This epitope is shown in FIG. 1, andincludes residues from about 541 to about 599, inclusive, in the aminoacid sequence of ErbB2 extracellular domain.

The “epitope 7C2/7F3” is the region at the N-terminus of theextracellular domain of ErbB2 to which the 7C2 and/or 7F3 antibodies(each deposited with the ATCC, see below) bind. To screen for antibodieswhich bind to the 7C2/7F3 epitope, a routine cross-blocking assay suchas that described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, E d Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to establish whether theantibody binds to the 7C2/7F3 epitope on ErbB2 (i.e. any one or more ofresidues in the region from about residue 22 to about residue 53 ofErbB2; SEQ ID NO: 2).

The term “induces cell death” or “capable of inducing cell death” refersto the ability of the antibody to make a viable cell become nonviable.The “cell” here is one which expresses the ErbB2 receptor, especiallywhere the cell overexpresses the ErbB2 receptor. A cell which“overexpresses” ErbB2 has significantly higher than normal ErbB2 levelscompared to a noncancerous cell of the same tissue type. Preferably, thecell is a cancer cell, e.g. a breast, ovarian, stomach, endometrial,salivary gland, lung, kidney, colon, thyroid, pancreatic or bladdercell. In vitro, the cell may be a SKBR3, BT474, Calu 3, MDA-MB-453,MDA-MB-361 or SKOV3 cell. Cell death in vitro may be determined in theabsence of complement and immune effector cells to distinguish celldeath induced by antibody dependent cellular cytotoxicity (ADCC) orcomplement dependent cytotoxicity (CDC). Thus, the assay for cell deathmay be performed using heat inactivated serum (i.e. in the absence ofcomplement) and in the absence of immune effector cells. To determinewhether the antibody is able to induce cell death, loss of membraneintegrity as evaluated by uptake of propidium iodide (PI), trypan blue(see Moore et al. Cytotechnology 17:1-11 [1995]) or 7AAD can be assessedrelative to untreated cells. Preferred cell death-inducing antibodiesare those which induce PI uptake in the “PI uptake assay in BT474cells”.

The phrase “induces apoptosis” or “capable of inducing apoptosis” refersto the ability of the antibody to induce programmed cell death asdetermined by binding of annexin V, fragmentation of DNA, cellshrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/orformation of membrane vesicles (called apoptotic bodies). The cell isone which overexpresses the ErbB2 receptor. Preferably the “cell” is atumor cell, e.g. a breast, ovarian, stomach, endometrial, salivarygland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Invitro, the cell may be a SKBR3, BT474, Calu 3 cell, MDA-MB-453,MDA-MB-361 or SKOV3 cell. Various methods are available for evaluatingthe cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNAfragmentation can be evaluated through DNA laddering as disclosed in theexample herein; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.Preferably, the antibody which induces apoptosis is one which results inabout 2 to 50 fold, preferably about 5 to 50 fold, and most preferablyabout 10 to 50 fold, induction of annexin binding relative to untreatedcell in an “annexin binding assay using BT474 cells” (see below).

Sometimes the pro-apoptotic antibody will be one which blocks HRGbinding/activation of the ErbB2/ErbB3 complex (e.g. 7F3 antibody). Inother situations, the antibody is one which does not significantly blockactivation of the ErbB2/ErbB3 receptor complex by HRG (e.g. 7C2).Further, the antibody may be one like 7C2 which, while inducingapoptosis, does not induce a large reduction in the percent of cells inS phase (e.g. one which only induces about 0-10% reduction in thepercent of these cells relative to control).

The antibody of interest may be one like 7C2 which binds specifically tohuman ErbB2 and does not significantly cross-react with other proteinssuch as those encoded by the erbB1, erbB3 and/or erbB4 genes. Sometimes,the antibody may not significantly cross-react with the rat neu protein,e.g., as described in Schecter et al. Nature 312:513 (1984) and Drebinet al., Nature 312:545-548 (1984). In such embodiments, the extent ofbinding of the antibody to these proteins (e.g., cell surface binding toendogenous receptor) will be less than about 10% as determined byfluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA).

“Heregulin” (HRG) when used herein refers to a polypeptide whichactivates the ErbB2-ErbB3 and ErbB2-ErbB4 protein complexes (i.e.induces phosphorylation of tyrosine residues in the complex upon bindingthereto). Various heregulin polypeptides encompassed by this term aredisclosed in Holmes et al., Science, 256:1205-1210 (1992); WO 92/20798;Wen et al., Mol. Cell. Biol., 14(3):1909-1919 (1994); and Marchionni etal., Nature, 362:312-318 (1993), for example. The term includesbiologically active fragments and/or variants of a naturally occurringHRG polypeptide, such as an EGF-like domain fragment thereof (e.g.HRGβ1₁₇₇₋₂₄₄).

The “ErbB2-ErbB3 protein complex” and “ErbB2-ErbB4 protein complex” arenoncovalently associated oligomers of the ErbB2 receptor and the ErbB3receptor or ErbB4 receptor, respectively. The complexes form when a cellexpressing both of these receptors is exposed to HRG and can be isolatedby immunoprecipitation and analyzed by SDS-PAGE as described inSliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994).

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework region (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting aβ-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the β-sheet structure.The CDRs in each chain are held together in close proximity by the FRsand, with the CDRs from the other chain, contribute to the formation ofthe antigen-binding site of antibodies (see Kabat et al., NIH Publ. No.91-3242, Vol. 1, pages 647-669 [1991]). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody dependent cellular cytotoxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these maybe further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “antibody” is used in the broadest sense and specificallycovers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10):1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementaritydetermining region (CDR) of the recipient are replaced by residues froma CDR of a non-human species (donor antibody) such as mouse, rat orrabbit having the desired specificity, affinity, and capacity. In someinstances, framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues which are found neither in therecipient antibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and maximize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDRs correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature, 321:522-525 (1986); Reichmannet al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992). The humanized antibody includes a PRIMATIZED™ antibodywherein the antigen-binding region of the antibody is derived from anantibody produced by immunizing macaque monkeys with the antigen ofinterest.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

A “disorder” is any condition that would benefit from treatment with theanti-ErbB2 antibody. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose themammal to the disorder in question. Non-limiting examples of disordersto be treated herein include benign and malignant tumors; leukemias andlymphoid malignancies; neuronal, glial, astrocytal, hypothalamic andother glandular, macrophagal, epithelial, stromal and blastocoelicdisorders; and inflammatory, angiogenic and immunologic disorders.

The term “therapeutically effective amount” is used to refer to anamount having antiproliferative effect. Preferably, the therapeuticallyeffective amount has apoptotic activity, or is capable of inducing celldeath, and preferably death of benign or malignant tumor cells, inparticular cancer cells. Efficacy can be measured in conventional ways,depending on the condition to be treated. For cancer therapy, efficacycan, for example, be measured by assessing the time to diseaseprogression (TTP), or determining the response rates (RR) (see Example1, below). Therapeutically effective amount also refers to a targetserum concentration, such as a trough serum concentration, that has beenshown to be effective in suppressing disease symptoms when maintainedfor a period of time.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidaniine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE®, Rhône-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included in this definition areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially anErbB2-overexpressing cancer cell either in vitro or in vivo. Thus, thegrowth inhibitory agent is one which significantly reduces thepercentage of ErbB2 overexpressing cells in S phase. Examples of growthinhibitory agents include agents that block cell cycle progression (at aplace other than S phase), such as agents that induce G1 arrest andM-phase arrest. Classical M-phase blockers include the vincas(vincristine and vinblastine), TAXOL®, and topo II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (W B Saunders:Philadelphia, 1995), especially p. 13. The 4D5 antibody (and functionalequivalents thereof) can also be employed for this purpose.

“Doxorubicin” is an athracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

By “solid phase” is meant a non-aqueous matrix to which the antibodiesused in accordance with the present invention can adhere. Examples ofsolid phases encompassed herein include those formed partially orentirely of glass (e.g.,controlled pore glass), polysaccharides (e.g.,agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.In certain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g.,an affinity chromatography column). This term also includesa discontinuous solid phase of discrete particles, such as thosedescribed in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the anti-ErbB2 antibodies disclosed herein and, optionally, achemotherapeutic agent) to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The term “serum concentration,” “serum drug concentration,” or “serumHERCEPTIN® anti-ErbB2 antibody concentration” refers to theconcentration of a drug, such as HERCEPTIN® anti-ErbB2 antibody, in theblood serum of an animal or human patient being treated with the drug.Serum concentration of HERCEPTIN® anti-ErbB2 antibody, for example, ispreferably determined by immunoassay. Preferably, the immunoassay is anELISA according to the procedure disclosed herein.

The term “peak serum concentration” refers to the maximal serum drugconcentration shortly after delivery of the drug into the animal orhuman patient, after the drug has been distributed throughout the bloodsystem, but before significant tissue distribution, metabolism orexcretion of drug by the body has occurred.

The term “trough serum concentration” refers to the serum drugconcentration at a time after delivery of a previous dose andimmediately prior to delivery of the next subsequent dose of drug in aseries of doses. Generally, the trough serum concentration is a minimumsustained efficacious drug concentration in the series of drugadministrations. Also, the trough serum concentration is frequentlytargeted as a minimum serum concentration for efficacy because itrepresents the serum concentration at which another dose of drug is tobe administered as part of the treatment regimen. If the delivery ofdrug is by intravenous administration, the trough serum concentration ismost preferably attained within 1 day of a front loading initial drugdelivery. If the delivery of drug is by subcutaneous administration, thepeak serum concentration is preferably attained in 3 days or less.According to the invention, the trough serum concentration is preferablyattained in 4 weeks or less, preferably 3 weeks or less, more preferably2 weeks or less, most preferably in 1 week or less, including 1 day orless using any of the drug delivery methods disclosed herein.

The term “intravenous infusion” refers to introduction of a drug intothe vein of an animal or human patient over a period of time greaterthan approximately 5 minutes, preferably between approximately 30 to 90minutes, although, according to the invention, intravenous infusion isalternatively administered for 10 hours or less.

The term “intravenous bolus” or “intravenous push” refers to drugadministration into a vein of an animal or human such that the bodyreceives the drug in approximately 15 minutes or less, preferably 5minutes or less.

The term “subcutaneous administration” refers to introduction of a drugunder the skin of an animal or human patient, preferable within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle. The pocket may be created by pinchingor drawing the skin up and away from underlying tissue.

The term “subcutaneous infusion” refers to introduction of a drug underthe skin of an animal or human patient, preferably within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle for a period of time including, but notlimited to, 30 minutes or less, or 90 minutes or less. Optionally, theinfusion may be made by subcutaneous implantation of a drug deliverypump implanted under the skin of the animal or human patient, whereinthe pump delivers a predetermined amount of drug for a predeterminedperiod of time, such as 30 minutes, 90 minutes, or a time periodspanning the length of the treatment regimen.

The term “subcutaneous bolus” refers to drug administration beneath theskin of an animal or human patient, where bolus drug delivery ispreferably less than approximately 15 minutes, more preferably less than5 minutes, and most preferably less than 60 seconds. Administration ispreferably within a pocket between the skin and underlying tissue, wherethe pocket is created, for example, by pinching or drawing the skin upand away from underlying tissue.

The term “front loading” when referring to drug administration is meantto describe an initially higher dose followed by the same or lower dosesat intervals. The initial higher dose or doses are meant to more rapidlyincrease the animal or human patient's serum drug concentration to anefficacious target serum concentration. According to the presentinvention, front loading is achieved by an initial dose or dosesdelivered over three weeks or less that causes the animal's or patient'sserum concentration to reach a target serum trough concentration.Preferably, the initial front loading dose or series of doses isadministered in two weeks or less, more preferably in 1 week or less,including 1 day or less. Most preferably, where the initial dose is asingle dose and is not followed by a subsequent maintenance dose for atleast 1 week, the initial dose is administered in 1 day or less. Wherethe initial dose is a series of doses, each dose is separated by atleast 3 hours, but not more than 3 weeks or less, preferably 2 weeks orless, more preferably 1 week or less, most preferably 1 day or less. Toavoid adverse immune reaction to an antibody drug such as an anti-ErbB2antibody (e.g., HERCEPTIN® anti-ErbB2 antibody) in an animal or patientwho has not previously been treated with the antibody, it may bepreferable to deliver initial doses of the antibody by intravenousinfusion. The present invention includes front loading drug delivery ofinitial and maintenance doses by infusion or bolus administration,intravenously or subcutaneously.

Published information related to anti-ErbB2 antibodies includes thefollowing issued patents and published applications: PCT/US89/0005 1,published Jan. 5, 1989; PCT/US90/02697, published May 18, 1990; EU0474727 issued Jul. 23, 1997; DE 69031120.6, issued Jul. 23, 1997;PCT/US97/18385, published Oct. 9, 1997; SA 97/9185, issued Oct. 14,1997; U.S. Pat. No. 5,677,171, issued Oct. 14, 1997; U.S. Pat. No.5,720,937, issued Feb. 24, 1998; U.S. Pat. No. 5,720,954, issued Feb.24, 1998; U.S. Pat. No. 5,725,856, issued Mar. 10, 1998; U.S. Pat. No.5,770,195, issued Jun. 23, 1998; U.S. Pat. No. 5,772,997, issued Jun.30, 1998; PCT/US98/2626, published Dec. 10, 1998; and PCT/US99/06673,published Mar. 26, 1999, each of which patents and publications isherein incorporated by reference in its entirety.

II. Production of anti-ErbB2 Antibodies

A description follows as to exemplary techniques for the production ofthe antibodies used in accordance with the present invention. The ErbB2antigen to be used for production of antibodies may be, e.g., a solubleform of the extracellular domain of ErbB2 or a portion thereof,containing the desired epitope. Alternatively, cells expressing ErbB2 attheir cell surface (e.g. NIH-3T3 cells transformed to overexpress ErbB2;or a carcinoma cell line such as SKBR3 cells, see Stancovski et al.,PNAS (USA) 88:8691-8695 [1991]) can be used to generate antibodies.Other forms of ErbB2 useful for generating antibodies will be apparentto those skilled in the art.

(i) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(ii) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 [Academic Press, 1986]).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63[Marcel Dekker, Inc., New York, 1987]).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103[Academic Press, 1986]). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 [1992]), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 [1993]). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl Acad. Sci. USA, 81:6851 [1984]), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iii) Humanized and Human Antibodies

Methods for humanizing non-human antibodies are well known in the art.Preferably, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al. Science, 239:1534-1536 [1988]), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901[1987]). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 [1993]).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal. Year in Immuno., 7:33 (1993). Human antibodies can also be derivedfrom phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581-597 [1991]).

(iv) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 [1985]). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10: 163-167 [1992]). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185.

(v) Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the ErbB2 protein. For example, onearm may bind an epitope in Domain 1 of ErbB2 such as the 7C2/7F3epitope, the other may bind a different ErbB2 epitope, e.g. the 4D5epitope. Other such antibodies may combine an ErbB2 binding site withbinding site(s) for EGFR, ErbB3 and/or ErbB4. Alternatively, ananti-ErbB2 arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 orCD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII(CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms tothe ErbB2-expressing cell. Bispecific antibodies may also be used tolocalize cytotoxic agents to cells which express ErbB2. These antibodiespossess an ErbB2-binding arm and an arm which binds the cytotoxic agent(e.g. saporin, anti-interferon-α, vinca alkaloid, ricin A chain,methotrexate or radioactive isotope hapten). Bispecific antibodies canbe prepared as full length antibodies or antibody fragments (e.g.F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 [1983]). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the C_(H)3domain of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991).

(vi) Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above. Thoseantibodies having the characteristics described herein are selected.

To select for antibodies which induce cell death, loss of membraneintegrity as indicated by, e.g., PI, trypan blue or 7AAD uptake isassessed relative to control. The preferred assay is the “PI uptakeassay using BT474 cells”. According to this assay, BT474 cells (whichcan be obtained from the American Type Culture Collection [Rockville,Md.]) are cultured in Dulbecco's Modified Eagle Medium (D-MEM):Ham'sF-12 (50:50) supplemented with 10% heat-inactivated FBS (Hyclone) and 2mM L-glutamine. (Thus, the assay is performed in the absence ofcomplement and immune effector cells). The BT474 cells are seeded at adensity of 3×10⁶ per dish in 100×20 mm dishes and allowed to attachovernight. The medium is then removed and replaced with fresh mediumalone or medium containing 10 μg/ml of the appropriate MAb. The cellsare incubated for a 3 day time period. Following each treatment,monolayers are washed with PBS and detached by trypsinization. Cells arethen centrifuged at 1200 rpm for 5 minutes at 4° C., the pelletresuspended in 3 ml ice cold Ca²⁺ binding buffer (10 mM Hepes, pH 7.4,140 mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mm strainer-capped12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal ofcell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzedusing a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software(Becton Dickinson). Those antibodies which induce statisticallysignificant levels of cell death as determined by PI uptake areselected.

In order to select for antibodies which induce apoptosis, an “annexinbinding assay using BT474 cells” is available. The BT474 cells arecultured and seeded in dishes as discussed in the preceding paragraph.The medium is then removed and replaced with fresh medium alone ormedium containing 10 μg/ml of the MAb. Following a three day incubationperiod, monolayers are washed with PBS and detached by trypsinization.Cells are then centrifuged, resuspended in Ca²⁺ binding buffer andaliquoted into tubes as discussed above for the cell death assay. Tubesthen receive labeled annexin (e.g. annexin V-FTIC) (1 μg/ml). Samplesmay be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™CellQuest software (Becton Dickinson). Those antibodies which inducestatistically significant levels of annexin binding relative to controlare selected as apoptosis-inducing antibodies.

In addition to the annexin binding assay, a “DNA staining assay usingBT474 cells” is available. In order to perform this assay, BT474 cellswhich have been treated with the antibody of interest as described inthe preceding two paragraphs are incubated with 9 μg/ml HOECHST 33342™for 2 hr at 37° C., then analyzed on an EPICS ELITE™ flow cytometer(Coulter Corporation) using MODFIT LT™ software (Verity Software House).Antibodies which induce a change in the percentage of apoptotic cellswhich is 2 fold or greater (and preferably 3 fold or greater) thanuntreated cells (up to 100% apoptotic cells) may be selected aspro-apoptotic antibodies using this assay.

To screen for antibodies which bind to an epitope on ErbB2 bound by anantibody of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed by methods known in theart.

To identify anti-ErbB2 antibodies which inhibit growth of SKBR3 cells incell culture by 50-100%, the SKBR3 assay described in WO 89/06692 can beperformed. According to this assay, SKBR3 cells are grown in a 1:1mixture of F12 and DMEM medium supplemented with 10% fetal bovine serum,glutamine and penicillinstreptomycin. The SKBR3 cells are plated at20,000 cells in a 35 mm cell culture dish (2 mls/35 mm dish). 2.5 μg/mlof the anti-ErbB2 antibody is added per dish. After six days, the numberof cells, compared to untreated cells are counted using an electronicCOULTER™ cell counter. Those antibodies which inhibit growth of theSKBR3 cells by 50-100% are selected for combination with the apoptoticantibodies as desired.

(vii) Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance the effectiveness of the antibodyin treating cancer, for example. For example, cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992)and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodieswith enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch 53:2560-2565 (1993). Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al. Anti-CancerDrug Design 3:219-230 (1989).

(viii) Immunoconjugates

The invention also pertains to immunoconjugates comprising the antibodydescribed herein conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g. an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated anti-ErbB2 antibodies. Examples include ²¹²Bi, ¹³¹I,¹³¹In, ⁹⁰Y and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as his(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide).

(ix) Immunoliposomes

The anti-ErbB2 antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al., J. National Cancer Inst. 81(19)1484 (1989).

(x) Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see WO 81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature 328:457-458 [1987]). Antibody-abzyme conjugates can be prepared as describedherein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the anti-ErbB2antibodies by techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 [1984]).

(xi) Antibody-Salvage Receptor Binding Epitope Fusions

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. In this case, it may be desirable to modifythe antibody fragment in order to increase its serum half life. This maybe achieved, for example, by incorporation of a salvage receptor bindingepitope into the antibody fragment (e.g. by mutation of the appropriateregion in the antibody fragment or by incorporating the epitope into apeptide tag that is then fused to the antibody fragment at either end orin the middle, e.g. by DNA or peptide synthesis).

A systematic method for preparing such an antibody variant having anincreased in vivo half-life comprises several steps. The first involvesidentifying the sequence and conformation of a salvage receptor bindingepitope of an Fc region of an IgG molecule. Once this epitope isidentified, the sequence of the antibody of interest is modified toinclude the sequence and conformation of the identified binding epitope.After the sequence is mutated, the antibody variant is tested to see ifit has a longer in vivo half-life than that of the original antibody. Ifthe antibody variant does not have a longer in vivo half-life upontesting, its sequence is further altered to include the sequence andconformation of the identified binding epitope. The altered antibody istested for longer in vivo half-life, and this process is continued untila molecule is obtained that exhibits a longer in vivo half-life.

The salvage receptor binding epitope being thus incorporated into theantibody of interest is any suitable such epitope as defined above, andits nature will depend, e.g., on the type of antibody being modified.The transfer is made such that the antibody of interest still possessesthe biological activities described herein.

The epitope preferably constitutes a region wherein any one or moreamino acid residues from one or two loops of a Fc domain are transferredto an analogous position of the antibody fragment. Even more preferably,three or more residues from one or two loops of the Fc domain aretransferred. Still more preferred, the epitope is taken from the CH2domain of the Fc region (e.g., of an IgG) and transferred to the CH1,CH3, or V_(H) region, or more than one such region, of the antibody.Alternatively, the epitope is taken from the CH2 domain of the Fc regionand transferred to the C_(L) region or V_(L) region, or both, of theantibody fragment.

In one most preferred embodiment, the salvage receptor binding epitopecomprises the sequence (5′ to 3′): PKNSSMISNTP (SEQ ID NO:3), andoptionally further comprises a sequence selected from the groupconsisting of HQSLGTQ (SEQ ID NO:4), HQNLSDGK (SEQ ID NO:5), HQNISDGK(SEQ ID NO:6), or VISSHLGQ (SEQ ID NO:7), particularly where theantibody fragment is a Fab or F(ab′)₂. In another most preferredembodiment, the salvage receptor binding epitope is a polypeptidecontaining the sequence(s)(5′ to 3′): HQNLSDGK (SEQ ID NO:5), HQNISDGK(SEQ ID NO:6), or VISSHLGQ (SEQ ID NO:7) and the sequence: PKNSSMISNTP(SEQ ID NO:3).

(xii) Purification of anti-ErbB2 Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems arepreferably first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 [1983]). G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 [1986]). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g. from about 0-0.25M salt).

III. Determination of anti-ErbB2 Antibody Concentration in Serum

The following non-limiting assay is useful for determining the presenceof and to quantitate the amount of specific rhuMAb HER2 (humanizedanti-p185^(HER2) monoclonal antibody, including HERCEPTIN® anti-ErbB2antibody) in a body fluid of a mammal including, but not limited to,serum, amniotic fluid, milk, umbilical cord serum, ocular aqueous andvitreous liquids, and ocular vitreous gel.

Plate Binding Activity Assay for rhuMAb HER2 (Humanized Anti-p185^(HER2)Monoclonal Antibody

The method of assaying rhuMAb HER2 described herein is meant as anexample of such a method and is not meant to be limiting. A standardizedpreparation of rhuMAb HER2 (Genentech, Inc., South San Francisco,Calif.), controls, and serum samples were diluted with Assay Diluent(PBS/0.5% BSA/0.05% Polysorbate 20/0.01% Thimerosal). The dilutions ofstandardized rhuMAb HER2 were prepared to span a range of concentrationsuseful for a standard curve. The samples were diluted to fall within thestandard curve.

An aliquot of Coat Antigen in Coating buffer (recombinant p185^(HER2)(Genentech, Inc.) in 0.05 M sodium carbonate buffer) was added to eachwell of a microtiter plate and incubated at 2-8° C. for 12-72 hours. Thecoating solution was removed and each well was washed six times withwater, then blotted to remove excess water.

An aliquot of Assay Diluent was added to each well and incubated for 1-2hours at ambient temperature with agitation. The wells were washed as inthe previous step.

Aliquots of diluted standard, control and sample solutions were added tothe wells and incubated at ambient temperature for 1 hour with agitationto allow binding of the antibody to the coating antigen. The wells arewashed again with water as in previous steps.

Horse radish peroxidase-conjugate (HRP-conjugate, Goat anti-human IgG Fcconjugated to horseradish peroxidase; Organon Teknika catalog #55253 orequivalent) was diluted with Assay Diluent to yield an appropriateoptical density range between the highest and lowest standards. Analiquot of the HRP-conjugate solution was added to each well andincubated at ambient temperature for I hour with agitation. The wellswere washed with water as in previous steps.

An aliquot of Substrate Solution (o-phenylenediamine (OPD) 5 mg tablet(Sigma P6912 or equivalent) in 12.5 ml 4 mM H₂O₂ in PBS) was added toeach well and incubated for a sufficient period of time (approximately8-10 minutes) in the dark at ambient temperature to allow colordevelopment. The reaction was stopped with an aliquot of 4.5 N sulfuricacid. Optical density was read at 490-492 nm for detection absorbanceand 405 nm for reference absorbance. The standard curve data are plottedand the results for the controls and samples are determined from thestandard curve.

IV. Pharmaceutical Formulations

Therapeutic formulations of the antibodies used in accordance with thepresent invention are prepared for storage by mixing an antibody havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16 th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Preferred lyophilized anti-ErbB2 antibodyformulations are described in WO 97/04801, expressly incorporated hereinbe reference.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide antibodies whichbind to EGFR, ErbB2 (e.g. an antibody which binds a different epitope onErbB2), ErbB3, ErbB4, or vascular endothelial growth factor (VEGF) inthe one formulation. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine or growth inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16 th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

V. Treatment with the Anti-ErbB2 Antibodies

It is contemplated that, according to the present invention, theanti-ErbB2 antibodies may be used to treat various conditionscharacterized by overexpression and/or activation of the ErbB2 receptor.Exemplary conditions or disorders include benign or malignant tumors(e.g. renal, liver, kidney, bladder, breast, gastric, ovarian,colorectal, prostate, pancreatic, lung, vulval, thyroid, hepaticcarcinomas; sarcomas; glioblastomas; and various head and neck tumors);leukemias and lymphoid malignancies; other disorders such as neuronal,glial, astrocytal, hypothalamic and other glandular, macrophagal,epithelial, stromal and blastocoelic disorders; and inflammatory,angiogenic and immunologic disorders.

The antibodies of the invention are administered to a human patient, inaccord with known methods, such as intravenous administration as a bolusor by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Intravenous or subcutaneous administration of the antibody is preferred.

The treatment of the present invention involves the administration of ananti-ErbB2 antibody to an animal or human patient, followed at intervalsby subsequent doses of equal or smaller doses such that a target serumconcentration is achieved and maintained during treatment. Preferably,maintenance doses are delivered by bolus delivery, preferably bysubcutaneous bolus administration, making treatment convenient andcost-effective for the patient and health care professionals.

Where combined administration of a chemotherapeutic agent (other than anantracycline) is desired, the combined administration includescoadministration, using separate formulations or a single pharmaceuticalformulation, and consecutive administration in either order, whereinpreferably there is a time period while both (or all) active agentssimultaneously exert their biological activities. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede,or follow administration of the antibody or may be given simultaneouslytherewith. The antibody may be combined with an anti-estrogen compoundsuch as tamoxifen or an anti-progesterone such as onapristone (see, EP616 812) in dosages known for such molecules.

It maybe desirable to also administer antibodies against other tumorassociated antigens, such as antibodies which bind to the EGFR, ErbB3,ErbB4, or vascular endothelial growth factor (VEGF). Alternatively, oradditionally, two or more anti-ErbB2 antibodies may be co-administeredto the patient. Sometimes, it may be beneficial to also administer oneor more cytokines to the patient. The ErbB2 antibody may beco-administered with a growth inhibitory agent. For example, the growthinhibitory agent may be administered first, followed by the ErbB2antibody. However, simultaneous administration, or administration of theErbB2 antibody first is also contemplated. Suitable dosages for thegrowth inhibitory agent are those presently used and may be lowered dueto the combined action (synergy) of the growth inhibitory agent andanti-ErbB2 antibody.

In addition to the above therapeutic regimens, the patient may besubjected to surgical removal of cancer cells and/or radiation therapy.

For the prevention or treatment of disease, the appropriate dosage ofanti-ErbB2 antibody will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether theantibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibody issuitably administered to the patient at one time or over a series oftreatments. Where the treatment involves a series of treatments, theinitial dose or initial doses are followed at daily or weekly intervalsby maintenance doses. Each maintenance dose provides the same or asmaller amount of antibody compared to the amount of antibodyadministered in the initial dose or doses.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. The progress ofthis therapy is easily monitored by conventional techniques and assays.

According to the invention, dosage regimens may include an initial doseof anti-ErbB2 of 6 mg/kg, 8 mg/kg, or 12 mg/kg delivered by intravenousor subcutaneous infusion, followed by subsequent weekly maintenancedoses of 2 mg/kg by intravenous infusion, intravenous bolus injection,subcutaneous infusion, or subcutaneous bolus injection. Where theantibody is well-tolerated by the patient, the time of infusion may bereduced.

Alternatively, the invention includes an initial dose of 12 mg/kganti-ErbB2 antibody, followed by subsequent maintenance doses of 6 mg/kgonce per 3 weeks.

Another dosage regimen involves an initial dose of 8 mg/kg anti-ErbB2antibody, followed by 6 mg/kg once per 3 weeks.

Still another dosage regimen involves an initial dose of 8 mg/kganti-ErbB2 antibody, followed by subsequent maintenance doses of 8 mg/kgonce per week or 8 mg/kg once every 2 to 3 weeks.

As an alternative regimen, initial doses of 4 mg/kg anti-ErbB2 antibodymay be administered on each of days 1, 2 and 3, followed by subsequentmaintenance doses of 6 mg/kg once per 3 weeks.

An additional regimen involves an initial dose of 4 mg/kg anti-ErbB2antibody, followed by subsequent maintenance doses of 2 mg/kg twice perweek, wherein the maintenance doses are separated by 3 days.

Alternatively, the invention may include a cycle of dosing in whichdelivery of anti-ErbB2 antibody is 2-3 times per week for 3 weeks. The 3week cycle is preferably repeated as necessary to achieve suppression ofdisease symptoms.

The invention further includes a cyclic dosage regimen in which deliveryof anti-ErbB2 antibody is daily for 5 days. According to the invention,the cycle is preferably repeated as necessary to achieve suppression ofdisease symptoms. Further information about suitable dosages is providedin the Examples below.

VI. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container, alabel and a package insert. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition which is effective for treating the condition and may have asterile access port (for example, the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is ananti-ErbB2 antibody. The label on, or associated with, the containerindicates that the composition is used for treating the condition ofchoice. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes. In addition, the article of manufacture may comprise a packageinserts with instructions for use, including, e.g., a warning that thecomposition is not to be used in combination with anthacycline-typechemotherapeutic agent, e.g. doxorubicin or epirubicin.

Deposit of Materials

The following hybridoma cell lines have been deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., USA(ATCC):

Antibody Designation ATCC No. Deposit Date 7C2 ATCC HB-12215 Oct. 17,1996 7F3 ATCC HB-12216 Oct. 17, 1996 4D5 ATCC CRT 10463 May 24, 1990 2C4ATCC HB-12697 Apr. 8, 1999

Further details of the invention are illustrated by the followingnon-limiting Examples.

EXAMPLES Example 1 Preparation and Efficacy of HERCEPTIN® Anti-ErbB2Antibody Materials and Methods

Anti-ErbB2 monoclonal antibody The anti-ErbB2 IgG₁κ murine monoclonalantibody 4D5, specific for the extracellular domain of ErbB2, wasproduced as described in Fendly et al., Cancer Research 5o:1550-1558(1990) and WO89/06692. Briefly, NIH 3T3/HER2-3₄₀₀ cells (expressingapproximately 1×10⁵ ErbB2 molecules/cell) produced as described inHudziak et al., Proc. Natl. Acad. Sci. (USA) 84:7159 (1987) wereharvested with phosphate buffered saline (PBS) containing 25 mM EDTA andused to immunize BALB/c mice. The mice were given injections i.p. of 10⁷cells in 0.5 ml PBS on weeks, 0, 2, 5 and 7. The mice with antisera thatimmunoprecipitated ³²P-labeled ErbB2 were given i.p. injections of awheat germ agglutinin-Sepharose (WGA) purified ErbB2 membrane extract onweeks 9 and 13. This was followed by an i.v. injection of 0.1 ml of theErbB2 preparation and the splenocytes were fused with mouse myeloma lineX63-Ag8.653. Hybridoma supernatants were screened for ErbB2-binding byELISA and radioimmunoprecipitation. MOPC-21 (IgG1), (Cappell, Durham,N.C.), was used as an isotype-matched control.

The treatment was performed with a humanized version of the murine 4D5antibody (HERCEPTIN® anti-ErbB2 antibody). The humanized antibody wasengineered by inserting the complementarity determining regions of themurine 4D5 antibody into the framework of a consensus humanimmunoglobulin IgG₁ (IgG₁) (Carter et al., Proc. Natl. Acad. Sci. USA89:4285-4289 [1992]). The resulting humanized anti-ErbB2 monoclonalantibody has high affinity for p185^(HER2) (Dillohiation constant[K_(d)]=0.1 nmol/L), markedly inhibits, in vitro and in humanxenografts, the growth of breast cancer cells that contain high levelsof p185^(HER2), induces antibody-dependent cellular cytotoxicity (ADCC),and has been found clinically active, as a single agent, in patientswith ErbB2-overexpressing metastatic breast cancers that had receivedextensive prior therapy. HERCEPTIN® anti-ErbB2 antibody is produced by agenetically engineered Chinese Hamster Ovary (CHO) cell line, grown inlarge scale, that secretes the antibody into the culture medium. Theantibody is purified from the CHO culture media using standardchromatographic and filtration methods. Each lot of antibody used inthis study was assayed to verify identity, purity, and potency, as wellas to meet Food and Drug Administration requirements for sterility andsafety.

Eligibility Criteria Patients had to fulfill all of the followingcriteria to be eligible for study admission:

-   -   Metastatic breast cancer    -   Overexpression of the ErbB2 (HER2) oncogene (2+ to 3+ as        determined by immunohistochemistry or fluorescence in situ        hybridization (FISH). [Tumor expression of ErbB2 can be        determined by immunohistochemical analysis, as previously        described (Slamon et al., [1987] and [1989], supra), of a set of        thin sections prepared from the patient's paraffin-archived        tumor blocks. The primary detecting antibody used is murine 4D5        MAb, which has the same CDRs as the humanized antibody used for        the treatment. Tumors are considered to overexpress ErbB2 if at        least 25% of tumor cells exhibit characteristic membrane        staining for p185^(HER2)].    -   Bidimensionally measurable disease (including lytic bone        lesions) by radiographic means, physical examination, or        photographs

Measurable disease was defined as any mass reproducibly measurable intwo perpendicular diameters by physical examination, X-ray (plainfilms), computerized tomography (CT), magnetic resonance imaging (MRI),ultrasound, or photographs.

Osteoblastic metastases, pleural effusions, or ascites were notconsidered to be measurable. Measurable lesions must be at least 1 cm ingreatest dimension. Enumeration of evaluable sites of metastatic diseaseand number of lesions in an evaluable site (e.g. lung) had to berecorded on the appropriate Case Report Form (CRF). If a large number ofpulmonary or hepatic lesions were present, the six largest lesions persite were followed.

-   -   The ability to understand and willingness to sign a written        informed consent form    -   Women>18 years    -   Suitable candidates for receiving concomitant cytotoxic        chemotherapy as evidenced by screening laboratory assessments of        hematologic, renal, hepatic, and metabolic functions.

Exclusion Criteria Patients with any of the following were excluded fromstudy entry:

-   -   Prior cytotoxic chemotherapy for metastatic breast cancer    -   Patients may have received prior hormonal therapy (e.g.        tamoxifen) for metastatic disease or cytotoxic therapy in the        adjuvant setting.    -   Concomitant malignancy that has not been curatively treated    -   A performance status of <60% on the Kamofsky scale    -   Pregnant or nursing women; women of childbearing potential,        unless using effective contraception as determined by the        investigator    -   Bilateral breast cancer (either both primary tumors must have 2+        to 3+ HER2 overexpression, or the metastatic site must have 2+        to 3+ HER2 overexpression)    -   Use of investigational or unlicensed agents within 30 days prior        to study entry    -   Clinically unstable or untreated metastases to the brain (e.g.        requiring radiation therapy)

Based upon the foregoing criteria, 469 patients were chosen, andenrolled in the study. Half the patients (stratified by chemotherapy)were randomized to additionally receive the HERCEPTIN® anti-ErbB2antibody (see below).

Administration and Dosage

Anti-ErbB2 Antibody

On day 0, a 4 mg/kg dose of humanized anti-ErbB2 antibody (HERCEPTIN®,H) was administered intravenously, over a 90-minute period. Beginning onday 7, patients received weekly administration of 2 mg/kg antibody(i.v.) over a 90-minute period.

Chemotherapy

The patients received one of two chemotherapy regimens for a minimum ofsix cycles, provided their disease was not progressing: a)cyclophosphamide and doxorubicin or epirubicin (AC), if patients havenot received anthracycline therapy in the adjuvant setting, or b)paclitaxel (T, TAXOL®), if patients have received any anthracyclinetherapy in the adjuvant setting. The initial dose of the HERCEPTIN®anti-ErbB2 antibody preceded the first cycle of either chemotherapyregimen by 24 hours. Subsequent doses of the antibody were givenimmediately before chemotherapy administration, if the initial dose ofthe antibody was well tolerated. If the first dose of the antibody wasnot well tolerated, subsequent infusions continued to precedechemotherapy administration by 24 hours. Patients were permitted tocontinue receiving chemotherapy beyond six cycles if, in the opinion ofthe treating physician, they were continuing to receive treatmentbenefit.

Cyclophosphamide (600 mg/m²) was given either by iv push over a minimumperiod of 3 minutes or by infusion over a maximum period of 2 hours.

Doxorubicin (60 mg/m²) or epirubicin (75 mg/m²) were given either byslow iv push over a minimum period of 3-5 minutes or by infusion over amaximum period of 2 hours, according to institutional protocol.

Paciltaxel (TAXOL®) was given at a dose of 175 mg/m² over 3 hours byintravenous administration. All patients receiving paclitaxel werepremedicated with dexamethasone (or its equivalent) 20 mg×2,administered orally 12 and 6 hours prior to paclitaxel; diphenhydramine(or its equivalent) 50 mg, iv, administered 30 minutes prior topaclitaxel, and dimetidine (or another H₂ blocker) 300 mg, iv,administered 30 minutes prior to paclitaxel.

Response Criteria

Progressive Disease Objective evidence of an increase of 25% or more inany measurable lesion. Progressive disease also includes those instanceswhen new lesions have appeared. For bone lesions, progression is definedas a 25% increase in objective measurement by plain film, CT, MRI;symptomatic new lesions not due to fracture; or requirement forpalliative radiotherapy.

Complete Response Disappearance of all radiographically and/or visuallyapparent tumor for a minimum of 4 weeks. Skin and chest wall completeresponses had to be confirmed by biopsy.

Partial Response A reduction of at least 50% in the sum of the productsof the perpendicular diameters of all measurable lesions for a minimumperiod of 4 weeks. No new lesions may have appeared, nor may any lesionshave progressed in size.

Minor Response A reduction of 25% to 49% in the sum of the products ofthe perpendicular diameters of all measurable lesions. No new lesionsmay have appeared, nor may any lesions have progressed in size.

Stable Disease No change of greater than 25% in the size of measurablelesions. No lesions may have appeared.

Time to disease progression (TTP) was calculated from the beginning oftherapy to progression. Confidence limits for response rates werecalculated using the exact method for a single proportion. (Fleiss, J L,Statistical Methods for Rates and Proportions (ed. 2), New York, N.Y.,Wiley, 1981, pp 13-17).

Results

At a median follow-up of 10.5 months, assessments of time to diseaseprogression (TTP in months) and response rates (RR) showed a significantaugmentation of the chemotherapeutic effect by HERCEPTIN® anti-ErbB2antibody, without increase in overall severe adverse events (AE):

TABLE 1 HERCEPTIN ® Anti-ErbB2 Antibody Efficacy Enrolled TTP(months)RR(%) AE(%) CRx 234 5.5 36.2 66 CRx + H 235 8.6* 62.00** 69 AC 145 6.542.1 71 AC + H 146 9.0 64.9 68 T 89 4.2 25.0 59 T + H 89 7.1 57.3 70 *p< 0.001 by log-rank test; **p < 0.01 by X² test; CRx: chemotherapy; AC:anthracycline/cyclophosphamide treatment; H: HERCEPTIN ® anti-ErbB2antibody; T: TAXOL ®

A syndrome of myocardial dysfunction similar to that observed withanthracyclines was reported more commonly with a combined treatment ofAC+H (18% Grade ¾) than with AC alone (3%), T (0%), or T+H (2%).

These data indicate that the combination of anti-ErbB2 antibodytreatment with chemotherapy markedly increases the clinical benefit, asassessed by response rates and the evaluation of disease progression.However, due to the increased cardiac side-effects of doxorubicin orepirubicin, the combined use of anthracyclines with anti-ErbB2 antibodytherapy is contraindicated. The results, taking into account risk andbenefit, favor treatment with HERCEPTIN® anti-ErbB2 antibody andpaclitaxel (TAXOL®) where a combined treatment regimen is desired.

Example 2 Pharmacokinetic and Pharmacodynamic Properties of Anti-ErbB2Antibody (HERCEPTIN®)

HERCEPTIN® anti-ErbB2 antibody was administered by intravenous infusionto human patients selected according to the criteria provided inExample 1. An initial dose of 4 mg/kg HERCEPTIN® anti-ErbB2 antibody wasdelivered by intravenous infusion, followed by subsequent i.v. infusionsof 2 mg/kg HERCEPTIN® anti-ErbB2 antibody weekly for several weeks. Twohundred thirteen patients began this treatment regimen and serum drugconcentration was obtained beyond 8 weeks for fewer than 90 patients asselective discontinuation of patients with rapidly progressing diseaseoccurred. Of the 213 patients who began treatment, serum troughconcentration data were available for 80 patients at Week 12, for 77patients at Week 16, for 44 patients at Week 20, for 51 patients at Week24, for 25 patients at Week 28, for 23 patients at Week 32, and for 37patients at Week 36.

HERCEPTIN® Anti-ErbB2 Antibody Trough Serum Concentrations for Weeks0-36

The HERCEPTIN® anti-ErbB2 antibody trough serum concentrations (μg/ml,mean±SE) from Week 2 through Week 36 are plotted in FIG. 3 (darkcircles). The number of patients was fairly constant because data frompatients discontinued from the program due to rapidly progressingdisease were excluded from this analysis. Trough serum concentrationstended to increase through Week 12 and tended to plateau after thattime.

HERCEPTIN® Anti-ErbB2 Antibody Trough and Peak Serum Concentrations forWeeks 1-8

Some HERCEPTIN® anti-ErbB2 antibody serum concentration data wereavailable for 212 of the original 213 patients. Trough and peak serumconcentration data reflecting the first HERCEPTIN® anti-ErbB2 antibodyinfusion were available for 195 of the 212 patients. For the seventhinfusion, trough serum concentration data were available for 137/212patients and peak serum concentration data were available for 114/212patients. Table 2 presents a summary of statistics from trough and peakserum concentrations for the first 8 weeks of treatment. Peak sampleswere drawn shortly after the end of HERCEPTIN® anti-ErbB2 antibodyadministration; trough samples were drawn prior to the subsequent dose(i.e., 1 week later). Serum concentrations of HERCEPTIN® anti-ErbB2antibody were determined as disclosed herein.

TABLE 2 HERCEPTIN ® Anti-ErbB2 Antibody Trough and Peak SerumConcentrations for the First 8 Weeks of Treatment (μg/ml) Dose Number nMean SD Minimun Maximum Peak 1 195 100.3 35.2 30.7 274.6 Trough 195 25.012.7 0.16 60.7 Peak 2 190 74.3 31.3 20.8 307.9 Trough 167 30.4 16.0 0.274.4 Peak 3 167 75.3 26.8 16.1 194.8 Trough 179 33.7 17.9 0.2 98.2 Peak4 175 80.2 26.9 22.2 167 Trough 132 38.6 20.1 0.2 89.4 Peak 5 128 85.929.2 27.8 185.8 Trough 141 42.1 24.8 0.2 148.7 Peak 6 137 87.2 32.2 28.9218.1 Trough 115 43.2 24.0 0.2 109.9 Peak 7 114 89.7 32.5 16.3 187.8Trough 137 48.8 24.9 0.2 105.2 Peak 8 133 95.6 35.9 11.4 295.6

The data in Table 2 suggest that there was an increase in trough serumconcentration over time. Of the many patients studied, there were 18patients for whom the trough concentrations did not exceed 20 μg/ml fromWeek 2 through Week 8. A HERCEPTIN® anti-ErbB2 antibody trough serumconcentration of 20 μg/ml was nominally targeted for these studies basedon prior pharmacologic studies in animals and exploratory analyses inclinical trials.

Patient response status was evaluated relative to serum concentration ofHERCEPTIN® anti-ErbB2 antibody. For this purpose, mean serumconcentration (an average of troughs and peaks) was calculated forvarious times and patient response status (where the patient responsestatus was determined by an independent Response Evaluation Committee).The increase in serum concentration between Weeks 2 and 8 appeared to begreater in responders than in nonresponders, suggesting that there is arelationship between response status and HERCEPTIN® anti-ErbB2 antibodyserum concentration. A statistical analysis (analysis of variance) oftrough serum concentration values at Week 2 and an average of Weeks 7and 8 in relation to response status indicated a highly significantrelationship between response status and average trough of Weeks 7 and 8(p<0.001). The results indicated that there was a significant differencebetween the trough serum concentration (average troughs of Weeks 7 and8) in the responders and nonresponders: trough concentrations were 60±20μg/ml in the responders versus 44±25 μg/ml in the nonresponders(mean±SD). HER2 overexpression level and type of metastatic sites wereassociated with significant differences in trough serum concentrations.At Week 2, patients with 2+ HER2 overexpression had significantly highertrough serum concentrations (n=40, mean=28.8 μg/ml, SD=10.4) comparedwith patients with 3+ HER2 overexpression (n=155, mean=24.1 μg/ml,SD=13.1). This difference in the average trough serum concentrations forWeeks 7 and 8 was no longer statistically significant. Further, at Week2, patients with superficial disease had significantly higher troughserum concentrations (n=12, mean 34.1 μg/ml, SD=12.0) compared withpatients with visceral disease (n=183, mean=24.4 μg/ml, SD=12.6). Thisdifference in the average trough serum concentrations for Weeks 7 and 8was significant. These data indicate that the rise in trough serumconcentrations between Weeks 2 and 7/8 occurs for human patients withvarious disease profiles.

In a subsequent, similarly designed study, human breast cancer patientswere treated with a loading dose of 8 mg/kg followed by maintenancedoses of4 mg/kg weekly. The results of this preliminary human studyindicated that an 8 mg/kg load:4 mg/kg weekly maintenance regimen wasefficacious in reducing tumor volume in the patients.

The data disclosed in this Example indicate that front loading ofantibody, such that a target serum concentration is reached morequickly, may be associated with improved outcomes.

Example 3 I.V. Bolus Delivery and Subcutaneous Infusion of HERCEPTIN®Anti-ErbB2 Antibody Effectively Decrease Tumor Volume in the Mouse

The efficacy of infusion or bolus delivery of humanized anti-ErbB2antibody (HERCEPTIN®, see Example 1 for preparation), either byintravenous injection or subcutaneous injection, was examined. Thepurpose of the study was to ask whether subcutaneous delivery wasfeasible and whether the convenient subcutaneous bolus delivery wasuseful in treating metastatic breast cancer in animals inoculated with acell line that overexpresses the HER2 gene. The results, detailed below,show that i.v. and s.c. infusion and bolus delivery are feasibletreatment methodologies.

A study in a nude mouse xenograft model, which incorporates a humanbreast cancer cell line that naturally overexpresses the HER2 gene(BT-474M1, derived from BT-474 cells, ATCC Accession number HTB-20),comparing tumor volume as a function of i.v. bolus versus s.c. infusionwas performed as follows. In the first study athymic nude nu nu 7-9 weekold female mice were obtained from Taconic Inc (Germantown, N.Y.). Toinitiate tumor development, each mouse was inoculated subcutaneouslywith 3×10⁶ BT474M1 cells suspended in Matrigel™. When tumor nodulesreached a volume of approximately 100 mm³, animals were randomized to 4treatment groups. The groups were treated according to Table 3.

TABLE 3 Animal Groups and Doses for Comparison of I.V. Bolus and S.C.Infusion Group, Target Loading Dose, Serum Conc. Route of DoseMaintenance Antibody μg/ml Administration (mg/kg) Dose 1 - Control, 20IV LD and 2.20 0.250 mg/ml rhuMAb E25 SC infusion (infusate) 2 - LowDose SC  1 IV LD and 0.313 0.050 mg/ml rhuMAb HER2 SC infusion(infusate) 3 - High Dose SC 20 IV LD and 6.25  1.00 mg/ml rhuMAb HER2 SCinfusion (infusate) 4 - IV Multi-Dose 20 IV LD and MD 4.00    2 mg/kg/rhuMAb HER2 (trough) week (IV bolus) Serum Conc. = concentration inserum. LD = loading dose. MD = maintenance dose. Infusate concentrationwas calculated to achieve targeted serum concentration using Alzet ®osmotic minipumps (Alza Corp., Palo Alto, CA).

Animals were exposed to estrogen by subcutaneous sustained releaseestrogen pellet 9 days before the start of dosing to promote growth ofgrafted tumor cells. The animals were inoculated with the BT474M 1 cells8 days before the beginning of treatment and tumors were allowed togrow. The animals were then treated with nonrelevant antibody E25(non-specific for HER2 receptor, but a member of the monoclonal IgGclass) or test antibody HERCEPTIN® anti-ErbB2 anitbody as indicated inTable 3. The dosage levels were selected to achieve target serumconcentrations of HERCEPTIN®, either 1 μg/ml or 20 μg/ml, bysubcutaneous pump infusion or by i.v. bolus delivery. The study groupswere treated until day 35. The serum concentration of HERCEPTIN®anti-ErbB2 antibody was measured weekly (just prior to dosing for Group4) using 3 mice/group/time point. The anti-ErbB2 antibody concentrationwas determined according to the method disclosed herein involvingstandard techniques. Tumor volumes were measured two days before dosingbegan and twice per week from day 6 to day 35 in the study for whichdata is tabulated below. Tumors were measured in three dimensions andvolumes were expressed in mm³. Efficacy was determined by a statisticalcomparison (ANOVA) of tumor volumes of test animals relative tountreated control animals.

As shown in Table 4, below, treatment of the BT474M 1 tumor-bearing micewith HERCEPTIN® anti-ErbB2 antibody by the indicated dosage methodssignificantly inhibited the growth of the tumors. All HERCEPTIN®-treatedgroups showed similar inhibition of tumor growth relative to the controlgroup. No dose-response was observed.

TABLE 4 Comparison of S.C. Infusion and I.V. Bolus Delivery Tumor VolumeHERCEPTIN ® Tumor Volume (area under curve) Serum Conc. (mm³), Day 35,Day 6-Day 35 (μg/ml), Day 27, Treatment Group (n = 14) (n = 13) (n = 3)control s.c. 764 ± 700 5650 ± 4700 4.16 ± 1.94 infusion s.c. infusion80.6 ± 158  1610 ± 1250 2.11 ± 1.74 (low dose) s.c. infusion   31 ± 75.61440 ± 1140 22.1 ± 5.43 (high dose) i.v. bolus dose* 49.7 ± 95.7 2150 ±1480 21.7 ± 17.1** s.c. = subcutaneous delivery; i.v. = intravenousdelivery. *4.0 mg/kg Loading Dose and 2.0 mg/kg/week Maintenance Dose.**at predose (trough serum concentration immediately prior to amaintenance dose)

The results tabulated above indicate that maintenance of a serumconcentration of approximately 2 μg/ml was as effective as aconcentration of 20 μg/ml in this study. The results indicated thatdosing by subcutaneous infusion was as effective as intravenous bolusdosing and achieved similar trough serum concentrations. The resultsalso indicate that the dose levels studied are at the top of thedose-response curve in this model and that subcutaneous dosing iseffective in treating breast cancer tumors. Thus, subcutaneousadministration of maintenance doses is feasible as part of a HERCEPTIN®anti-ErbB2 antibody treatment regimen.

Example 4 I.V. Bolus and Subcutaneous Bolus Deliveries of HERCEPTIN®Anti-ErbB2 Antibody Effectively Decrease Tumor Volume in the Mouse

Subcutaneous bolus delivery is convenient and cost-effective for thepatient and health care professionals. The results of the studydisclosed in this example indicate that subcutaneous bolus delivery wasas effective as intravenous bolus delivery in reducing breast cell tumorsize in a mouse.

This study was set up as disclosed herein in Example 3 for thecomparison of intravenous bolus and subcutaneous infusion delivery. Asustained release estrogen implant was inserted subcutaneously one daybefore tumor cell innoculation as described in Example 3. Six days aftertumor cell innoculation, the initial tumor measurement was performed.Seven days after tumor cell innoculation, the first dose of controlantibody or HERCEPTIN® anti-ErbB2 antibody was delivered. The animalgroups, type of delivery, loading dose and maintenance doses areprovided in Table 4. Animals were dosed once weekly for 4 weeks.

TABLE 5 Animal Groups and Doses for Comparison of I.V. Bolus and S.C.Bolus Delivery Maintenance Route of Ad- Loading Dose Dose Groupministration (mg/kg) (mg/kg/week) n 1 - Control IV 8 4 10 rhuMAb E25 2 -rhuMAb HER2 IV 2 1 10 3 - rhuMAb HER2 IV 4 2 10 4 - rhuMAb HER2 IV 8 410 5 - rhuMAb HER2 SC 4 2 10 IV = intraveneous; SC = subcutaneous; n =number of animals per group.

The mice were treated according to the information in Table 4 and usingthe techniques disclosed in Example 3. The serum concentration ofHERCEPTIN® anti-ErbB2 antibody was measured weekly before each weeklyi.v. maintenance dose according to the procedure described herein andusing standard techniques. The control E25 antibody serum concentrationwas determined according to standard immunoassay techniques. Table 6shows the increase in HERCEPTIN® anti-ErbB2 antibody serumconcentrations with time.

TABLE 6 IV versus SC Bolus Delivery: Serum HERCEPTIN ® Anti-ErbB2Antibody Concentration Serum Concentration, μg/ml Day 0 Day 7 Day 14 Day21 Treatment Group Mean Mean Mean Mean (delivery, MD) (SD) (SD) (SD)(SD) 1 - Control rhu MAb E25 0 25.9 34.6 38.5 (IV, 4 mg/kg) (0) (8.29)(11.2) (14.4) 2 - rhu MAb HER2 0 4.96 8.55 8.05 (IV, 1 mg/kg) (0) (3.79)(5.83) (4.67) 3 - rhu MAb HER2 0 13.4 18.9 22.6 (IV, 2 mg/kg) (0) (9.24)(12.0) (9.21) 4 - rhu MAb HER2 0 29.6 37.7 46.2 (IV, 4 mg/kg) (0) (13.5)(14.4) (13.8) 5 - rhu MAb HER2 0 12.5 16.9 17.6 (SC, 2 mg/kg) (0) (7.33)(10.2) (10.7) n = 10 for time points Days 0, 7 and 14. N = 9 for Day 21.

Table 7 shows the relative efficacy of intravenous bolus delivery andsubcutaneous bolus delivery for Groups 1-5 having achieved the serumantibody concentrations presented in Table 6. For this study, efficacywas measured as a decrease in tumor volume. Tumor volume was measuredtwice weekly.

TABLE 7 Efficacy of HERCEPTIN ® Anti-ErbB2 Antibody Measured as a Changein Tumor Volume Comparing Intravenous Bolus and Subcutaneous BolusDelivery, Mean (SD) Treatment Tumor Tumor Vol. Day 6-Day 31* TumorGrowth Group Tumor Vol. Vol. Day Day 31, Area Under Curve Rate(Delivery, MD) Day 6, mm³ 28, mm³ mm³ Tumor Vol., mm³ on Log (TM + 1)1-IV Control 321 1530 1630 13600 0.0660 (190) (1040) (1170) (7230)(0.0200) 2-IV Herceptin 297 175 151 4690 −0.0505 1 mg/kg (130) (215)(188) (1400) (0.142) 3-IV Herceptin 269 75.7 73.6 3510 −0.0608 2 mg/kg(129) (92.4) (84.5) (1220) (0.110) 4-IV Herceptin 272 25.3 25.8 2880−0.0810 4 mg/kg (117) (75.9) (72.9) (1230) (0.0859) 5-SC Herceptin 26876.2 90.4 3230 −0.0304 2 mg/kg (117) (98.8) (105) (1440) (0.104) N = 10for each data point. TM = tumor measurement. IV = intravenous. SC =subcutaneous. MD = maintenance dose. Tumor Vol. = tumor volume, mm³.*Day 17 excluded due to measurement error. Tumor growth rate calculatedon Day 21-Day 31 Log (TM + 1). Area under the curve is the area beneatha plot of tumor volume versus time.

FIGS. 4A and 4B are graphical plots of changes in tumor volume overtime, some of which data is found in Table 7. FIG. 4A is a linear plotof tumor volume versus time. FIG. 4B is a semilogarithmic plot of thesame data, allowing the test points be viewed more clearly. The data inTable 7 and FIGS. 4A and 4B indicate that, although a dose-relatedresponse was not observed between HERCEPTIN-treated groups, dosing bysubcutaneous bolus was as effective as intravenous bolus dosing andachieved similar trough serum concentrations.

Example 5 Regimens for Intravenous and Subcutaneous Delivery ofAnti-ErbB2 Antibody

According to the invention, methods of anti-ErbB2 antibody (e.g.,HERCEPTIN®) delivery comprise greater front loading of the drug toachieve a target serum concentration in approximately 4 weeks or less,preferably 3 weeks or less, more preferably 2 weeks or less, and mostpreferably 1 week or less, including one day or less. According to theinvention, this initial dosing is followed by dosing that maintains thetarget serum concentration by subsequent doses of equal or smalleramount. An advantage of the methods of the invention is that themaintenance dosing may be less frequent and/or delivered by subcutaneousinjection, making the treatment regimens of the invention convenient andcost-effective for the patient and medical professionals administeringthe antibody. In addition, a subcutaneous maintenance dose regimen maybe interrupted by intravenous dosing (such as infusion) when thepatient's chemotherapy requires delivery of other drugs by intravenousinjection.

To test the following dosage regimens, human subjects are selectedaccording to the criteria disclosed in Example 1, above. The number ofinitial doses is one or more doses sufficient to achieve an efficacioustarget serum concentration in approximately 4 weeks or less, preferably3 weeks or less, more preferably 2 weeks or less, and most preferably 1week or less, including 1 day or less. The number of maintenance dosesmay be one or more doses sufficient to achieve suppression of diseasesymptoms, such as a decrease in tumor volume. The maintenance doses areequal to or smaller than the initial dose or doses, consistent with anobject of the invention of administering HERCEPTIN® anti-ErbB2 antibodyby regimens providing greater front loading. The specific drug deliveryregimens disclosed herein are representative of the invention and arenot meant to be limiting.

In one trial, an initial dose of 6 mg/kg, 8 mg/kg, or 12 mg/kg ofHERCEPTIN® anti-ErbB2 antibody is delivered to human patients byintravenous or subcutaneous injection. Initial doses (loading doses) aredelivered by intravenous infusion or bolus injection or preferablysubcutaneous bolus injection. Preferably a target trough serumconcentration of HERCEPTIN® anti-ErbB2 antibody of approximately 10-20μg/ml is achieved (averaged for all patients in the treatment group) andmaintained by subsequent doses of anti-ErbB2 antibody that are equal toor smaller than the initial dose. In one method, a target trough serumconcentration is achieved and maintained by once-per-week deliveries of2 mg/kg HERCEPTIN® anti-ErbB2 antibody by intravenous or subcutaneousinjection for at least eight weeks. Alternatively, for this or anydosage regimen disclosed herein, subcutaneous continuous infusion bysubcutaneous pump is used to delivery subsequent maintenance doses.

In another method, an initial (front loading) dose of 8 mg/kg HERCEPTIN®anti-ErbB2 antibody is delivered by intravenous injection (infusion orbolus injection) or by subcutaneous bolus injection. This is followed byintravenous bolus injections, intravenous infusion, subcutaneousinfusion, or subcutaneous bolus injection of 6 mg/kg at 3-week intervalsto maintain a trough serum concentration of approximately 10-20 μg/ml,averaged for an entire treatment group.

In another method, an initial (front loading) dose of 12 mg/kgHERCEPTIN® anti-ErbB2 antibody is delivered by intravenous injection(infusion or bolus injection) or by subcutaneous bolus injection. Thisis followed by intravenous bolus injections, intravenous infusion,subcutaneous infusion, or subcutaneous bolus injection of 6 mg/kg at3-week intervals to maintain a trough serum concentration ofapproximately 10-20 μg/ml.

In yet another method, an initial (front loading) dose of 8 mg/kgHERCEPTIN® anti-ErbB2 antibody is delivered by intravenous infusion orbolus injection, or preferably by subcutaneous bolus injection orinfusion. This is followed by administration of 8 mg/kg per week or 8mg/kg per 2-3 weeks to maintain a trough serum concentration ofHERCEPTIN® anti-ErbB2 antibody of approximately 10-20 μg/ml. Maintenancedoses are delivered by intravenous infusion or bolus injection, orpreferably by subcutaneous infusion or bolus injection.

In another method, the front loading initial dose is a series ofintravenous or subcutaneous injections, for example, one on each of days1, 2, and 3 of at least 1 mg/kg for each injection (where the amount ofanti-ErbB2 antibody delivered by the sum of initial injections is morethan 4 mg/kg), followed by maintenance doses of 6 mg/kg once each 3 weekinterval to maintain a target trough serum concentration (for example,approximately 10-20 μg/ml) of HERCEPTIN® anti-ErbB2 antibody. Themaintenance doses are delivered by intravenous infusion or bolusinjection or by subcutaneous infusion or subcutaneous bolus injection.

In yet another method, the front loading is by intravenous infusion ofat least 1 mg/kg, preferably 4 mg/kg on each of five consecutive days,followed by repeats of this cycle a sufficient number of times toachieve suppression of disease symptoms. Following the initial dose ordoses, subsequent doses may be delivered by subcutaneous infusion orbolus injection if tolerated by the patient. Such subcutaneous deliveryis convenient and cost-effective for the patient and administeringhealth care professionals.

In still another method, HERCEPTIN® anti-ErbB2 antibody is deliveredinitially as at least 2 intravenous infusions per week for three weeks,followed by repeats of this cycle to maintain an efficacious troughserum concentration of HERCEPTIN® anti-ErbB2 antibody. The dose is atleast 4 mg/kg of anti-ErbB2 antibody, preferably at least 5 mg/kg. Themaintenance drug deliveries may be intravenous or subcutaneous.

Where the animal or patient tolerates the antibody during and after aninitial dose, delivery of subsequent doses may be subcutaneous, therebyproviding greater convenience and cost-effectiveness for the patient andhealth care professionals.

In animal studies, an initial dose of more than 4 mg/kg, preferably morethan 5 mg/kg delivered by intravenous or subcutaneous injection, isfollowed by subcutaneous bolus injections of 2 mg/kg twice per week(separated by 3 days) to maintain a trough serum concentration ofapproximately 10-20 μg/ml. In addition, where the animal or patient isknown to tolerate the antibody, an initial dose of HERCEPTIN® anti-ErbB2antibody is optionally and preferably deliverable by subcutaneous bolusinjection followed by subcutaneous maintenance injections.

While target serum concentrations are disclosed herein for the purposeof comparing animal studies and human trials, target serumconcentrations in clinical uses may differ. The disclosure providedherein guides the user in selecting a front loading drug deliveryregimen that provides an efficacious target trough serum concentration.

The methods of the invention disclosed herein optionally include thedelivery of HERCEPTIN® anti-ErbB2 antibody in combination with achemotherapeutic agent (other than an anthrocycline derivative) toachieve suppression of disease symptoms. The chemotherapeutic agent maybe delivered with HERCEPTIN® anti-ErbB2 antibody or separately andaccording to a different dosing schedule. For example, subcutaneousdelivery of HERCEPTIN® anti-ErbB2 antibody with TAXOL® is included inthe invention. In addition, intravenous or subcutaneous injection of 8mg/kg HERCEPTIN® anti-ErbB2 antibody, followed by intravenous orsubcutaneous injection of 6 mg/kg HERCEPTIN® anti-ErbB2 antibody every 3weeks is administered in combination with a chemotherapeutic agent, suchas a taxoid (e.g. paclitaxel 175 mg/m2 every 3 weeks) or ananthracycline derivative (e.g. doxorubicin 60 mg/m2 or epirubicin 75mg/m2 every 3 weeks). Optionally, where an anthracycline derivative isadministered, a cardioprotectant (e.g. 600 mg/m2 cyclophosphamide every3 weeks) is also administered. In another combination therapy,anti-ErbB2 antibody is administered in a loading dose of more than 4mg/kg, preferably more than 5 mg/kg, and more preferably at least 8mg/kg. The loading dose is followed by maintenance doses of at least 2mg/kg weekly, preferably 6 mg/kg every 3 weeks. The combination therapyincludes administration of a taxoid during treatment with anti-ErbB2antibody. According to one embodiment of the invention, the taxoid ispaclitaxel and is administered at a dose of 70-100 mg/m²/week. Accordingto another embodiment of the invention, the taxoid is docetaxel and isadministered at a dose of 30-70 mg/m²/week.

Example 6 HERCEPTIN® Administered Intravenously Every Three Weeks inCombination with Paclitaxel

Currently, the recommended dose of HERCEPTIN® is 2 mg/kg once weekly.Patients will be administered HERCEPTIN® every three weeks instead ofweekly, along with paclitaxel (175 mg/m² every three weeks). Simulationof the proposed treatment regimen suggests that the trough serumconcentrations will be 17 mcg/ml, in the range (10-20 mcg/ml) of thetargeted trough serum concentrations from previous HERCEPTIN® IVclinical trials. After the first 12 patients the PK parameters will beassessed, if exposure is felt inadequate, then the dose will beincreased to 8 mg/kg every three weeks for the remaining 12 patients.

Inclusion Criteria

-   1) Females24 18 years of age-   2) Histologically confirmed ErbB2 over-expressing metastatic breast    cancer-   3) Patients who have been newly diagnosed with metastatic disease-   4) Have a Karnofsky performance status of 24 70%-   5) Give written informed consent prior to any study specific    screening procedures with the understanding that the patient has the    right to withdraw from the study at any time, without prejudice.

Exclusion Criteria

-   1) Pregnant or lactating women-   2) Women of childbearing potential unless (1) surgically sterile    or (2) using adequate measures of contraception such as oral    contraceptive, intra-uterine device or barrier method of    contraception in conjunction with spermicidal jelly.-   3) Clinical or radiologic evidence of CNS metastases.-   4) History of any significant cardiac disease-   5) LVEF≦50%-   6) No prior taxane therapy in any treatment setting.-   7) Any of the following abnormal baseline hematologic values:    -   Hb less than 9 g/dl    -   WBC less than 3.0×10⁹/l    -   Granulocytes less than 1.5×10⁹/l    -   Platelets less than 100×10⁹/l-   8) Any of the following abnormal baseline liver function tests:    -   Serum bilirubin greater than 1.5×ULN (upper normal limit)    -   ALT and/or AST greater than 2.5×ULN (greater than 4.0×ULN if        liver or bone metastasis)    -   Alkaline phosphatase greater than 2.5×ULN (greater than 4.0×ULN        if liver or bone metastasis)-   9) The following abnormal baseline renal function tests:    -   serum creatinine greater than 1.5×ULN-   10) History of other serious medical conditions that would preclude    patient participation in an investigational study.

HERCEPTIN® Loading dose and schedule: 8 mg/kg for first dose.Maintenance dose and schedule: 6 mg/kg every 3 weeks.

Paclitaxel—175 mg/m² IV every 3 weeks×6 cycles as a 3-hour infusion.

NOTE: On the first cycle of treatment, paclitaxel will be dosed 8 hoursprior to HERCEPTIN® to determine the PK of paclitaxel alone. HERCEPTIN®will be administered 8 hours post-paclitaxel for the 1^(st) cycle only.In subsequent treatment cycles, HERCEPTIN® will be administered prior topaclitaxel.

The total duration of this study is 18 weeks. Study subjects willreceive up to 6 total HERCEPTIN® doses. After the last subject hasreceived the last cycle of paclitaxel, data collection for safety andpharmacokinetic analysis will stop, and the study will close to protocolspecified treatment. Study subjects may continue to receive theHERCEPTIN® +/− paclitaxel at the discretion of the investigator.

It is believed that the above treatment regimen will be effective intreating metastatic breast cancer, despite the infrequency with whichHERCEPTIN® is administered to the patient.

While the particular aspects and embodiments of the invention as hereinshown and disclosed in detail is fully capable of obtaining the objectsand providing the advantages herein before stated, it is to beunderstood that it is merely illustrative of some of the presentlypreferred embodiments of the invention and that no limitations areintended to the details of methods and articles of manufacture shownother than as described in the appended claims. The disclosures of allcitations in the specification are expressly incorporated herein byreference.

1. A method for the treatment of a human patient diagnosed with cancercharacterized by overexpression of ErbB2 receptor, comprisingadministering an effective amount of an anti-ErbB2 antibody to the humanpatient, the method comprising: administering to the patient an initialdose of at least approximately 5 mg/kg of the anti-ErbB2 antibody; andadministering to the patient a plurality of subsequent doses of theantibody in an amount that is approximately the same or less than theinitial dose, wherein the subsequent doses are separated in time fromeach other by at least two weeks; and further comprising administeringan effective amount of a chemotherapeutic agent to the patient.
 2. Themethod of claim 1, wherein the initial dose is at least approximately 6mg/kg.
 3. The method of claim 2, wherein the initial dose is at leastapproximately 8 mg/kg.
 4. The method of claim 3, wherein the initialdose is at least approximately 12 mg/kg.
 5. The method of claim 1,wherein the subsequent doses are separated in time from each other by atleast three weeks.
 6. The method of claim 1, wherein the initial dose isadministered by intravenous injection, and wherein at least onesubsequent dose is administered by subcutaneous injection.
 7. The methodof claim 1, wherein the initial dose is administered by intravenousinjection, wherein at least two subsequent doses are administered, andwherein each subsequent dose is administered by a method selected fromthe group consisting of intravenous injection and subcutaneousinjection.
 8. The method of claim 1, wherein the initial dose and atleast one subsequent dose are administered by subcutaneous injection. 9.The method of claim 1, wherein the initial dose is selected from thegroup consisting of approximately 6 mg/kg, 8 mg/kg, or 12 mg/kg, whereinthe plurality of subsequent doses are at least approximately 2 mg/kg.10. The method of claim 9, wherein the plurality of subsequent doses areseparated in time from each other by at least three weeks.
 11. Themethod of claim 10, wherein the initial dose is approximately 8 mg/kg,and wherein at least one subsequent dose is approximately 6 mg/kg. 12.The method of claim 10, wherein the initial dose is approximately 12mg/kg, and wherein at least one subsequent dose is approximately 6mg/kg.
 13. The method of claim 9, wherein the initial dose isapproximately 8 mg/kg, and wherein at least one subsequent dose isapproximately 8 mg/kg.
 14. The method of claim 9, wherein the initialdose is approximately 8 mg/kg, wherein at least one subsequent dose is 8mg/kg, and wherein administration of the initial dose and subsequentdoses are separated in time by at least 2 weeks.
 15. The method of claim14, wherein the initial dose and subsequent doses are separated in timeby at least 3 weeks.
 16. The method of claim 1, wherein said cancer isselected from the group consisting of breast cancer, leukemia, squamouscell cancer, small-cell lung cancer, non-small cell lung cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, coloncancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.
 17. The method of claim 16, wherein said cancer is breastcancer.
 18. The method of claim 17, wherein said cancer is metastaticbreast carcinoma.
 19. The method of claim 1, wherein said antibody bindsto the extracellular domain of the ErbB2 receptor.
 20. The method ofclaim 19, wherein said antibody binds to epitope 4D5 within the ErbB2extracellular domain sequence.
 21. The method of claim 20, wherein saidantibody is a humanized 4D5 anti-ErbB2 antibody.
 22. The method of claim1, wherein the chemotherapeutic agent is a taxoid.
 23. The method ofclaim 22, wherein said taxoid is paclitaxel or docetaxel.
 24. The methodof claim 1, wherein the effective amount of the anti-ErbB2 antibody andthe effective amount of the chemotherapeutic agent as a combination islower than the sum of the effective amounts of said anti-ErbB2 antibodyand said chemotherapeutic agent, when administered individually, assingle agents.
 25. The method of claim 1, wherein the chemotherapeuticagent is an anthracycline.
 26. The method of claim 25, wherein theanthracycline is doxorubicin or epirubicin.
 27. The method of claim 25,wherein the method further comprises administration of acardioprotectant.
 28. The method of claim 1, wherein efficacy ismeasured by determining the time to disease progression or the responserate.
 29. A method for the treatment of a human patient diagnosed withcancer characterized by overexpression of ErbB2 receptor, comprisingadministering an effective amount of an anti-ErbB2 antibody to the humanpatient, the method comprising: administering to the patient an initialdose of the antibody, wherein the initial dose is a plurality of doses,wherein each of the plurality of initial doses is at least approximately1 mg/kg and is administered on at least 3 consecutive days, andadministering to the patient at least one subsequent dose of theantibody, wherein at least one subsequent dose is at least approximately6 mg/kg, and wherein administration of the last initial dose and thefirst subsequent and additional subsequent doses are separated in timeby at least 3 weeks, and further comprising administering an effectiveamount of a chemotherapeutic agent to the patient.
 30. A method for thetreatment of cancer in a human patient comprising administering to thepatient a first dose of an anti-ErbB2 antibody followed by two or moresubsequent doses of the antibody, wherein the subsequent doses areseparated from each other in time by at least about two weeks, andfurther comprising administering an effective amount of achemotherapeutic agent to the patient.
 31. The method of claim 30,wherein the first dose and a first subsequent dose are separated fromeach other in time by at least about three weeks.
 32. The method ofclaim 30, wherein the first dose and subsequent doses are each fromabout 2 mg/kg to about 16 mg/kg.
 33. The method of claim 32, wherein thefirst dose and subsequent doses are each from about 4 mg/kg to about 12mg/kg.
 34. The method of claim 33, wherein the first dose and subsequentdoses are each from about 6 mg/kg to about 12 mg/kg.
 35. The method ofclaim 30, wherein from about two to about ten subsequent doses of theantibody are administered to the patient.
 36. The method of claim 30,wherein the two or more subsequent doses are separated from each otherin time by at least about three weeks.
 37. The method of claim 30,wherein the two or more subsequent doses are each from about 2 mg/kg toabout 16 mg/kg.
 38. The method of claim 30, wherein the two or moresubsequent doses are each from about 4 mg/kg to about 12 mg/kg.
 39. Themethod of claim 30, wherein the two or more subsequent doses are eachfrom about 6 mg/kg to about 12 mg/kg.
 40. The method of claim 30,wherein the chemotherapeutic agent is a taxoid.